The Iron had not beaten the Blades on their own patch since their first League visit back in 1958, so when Paul Hayes fired home in first-half stoppage time the visiting fans were fantasising about an historic double after their 3-1 win at Glanford Park earlier this season.
And despite intense second-half pressure from Kevin Blackwell's side, they could not beat the impressive Joe Murphy in Scunthorpe's goal.
It meant just two points from a possible nine for the Blades – after trips to Doncaster Rovers and Cardiff City – and the boos that cascaded down on Blackwell from the Bramall Lane stands at the final whistle suggested even the most ardent Blade accepts their play-off hopes are over.
They trail sixth-placed Leicester City by seven points, having played a game more than the Foxes, and the Blades now need to win their remaining six matches just to stand a chance of gatecrashing the play-offs. It is a tall order that even Blackwell himself accepts is unlikely.
But the under-fire Blades boss insisted his management team had performed a "terrific feat" this season in keeping their injury-hit squad in contention for so long.
"If you're going to be manager of a high-profile club you have to take stick, rightly or wrongly," he said. "Anybody that saw the team there today realises that seven of them either aren't my players or aren't going to be here in six weeks' time.
"We're disappointed. The staff this year have been absolutely phenomenal to keep us where we are. I think (goalkeeper Steve Simonsen) Simmo was the 23rd debut in a season. When was the last time Sheffield United had five goalkeepers in a season never mind 23 loans?
"We've had 39 players play for the club this year. That leads us to be in the situation we're in, so to be up near the top with seven games to go has been a phenomenal achievement by the staff."
Blackwell believes this season has been one of his toughest yet, and feels he has done a good job.
"As one of my management roles this is one of the best jobs I've done," he said. "I could go on and on about the number of (loan) players who aren't my own, so it's been a terrific feat by my staff up to today to make sure we can perform.
"Temporary staff give you temporary performances and that means up and down, good and bad, and that's exactly what we've been like."
In a season dominated by injury problems for United – 22 players have made their debut in the red and white stripes – Blackwell was at least able to welcome Darius Henderson and Mark Yeates back after spells in the treatment room. There was also a place for Henri Camara on the substitutes bench.
United were indebted to a great tackle from Stephen Quinn on Donal McDermott as the Iron winger dashed into the home penalty area.
Quinn then popped up at the other end of the pitch, driving to the line before pulling a low, hard cross across goal which just evaded the onrushing Richard Cresswell. The former York City striker then turned provider twice, but Henderson and Yeates both failed to get their headers on target, while David Mirfin cleared off his line from Paul Connolly's shot.
At the other end there was a heart-stopping moment for goalkeeper Steve Simonsen, making his Bramall Lane debut after arriving on loan from Stoke City. Grant McCann's 25-yard free kick looked comfortable for Simonsen but he dropped the ball close to his line but there was no Iron player following up and the goalkeeper gratefully picked up the loose ball.
Simonsen then thwarted Garry Thompson in a one-on-one after the Scunthorpe striker pounced on a weak back pass from Kyle Bartley.
Yeates had a 25-yard free kick which flashed by Joe Murphy's left-hand post but it was Scunthorpe who took the lead within the four minutes of first-half stoppage time.
McDermott crossed from the right and former Barnsley striker Hayes snapped up the chance.
Blackwell looked to freshen up his attack early in the second half, Camara coming on for James Harper, but it was Yeates who came closest to finding a goal when his free kick was tipped over by Joe Murphy.
It proved to be the winger's last act as he was replaced by Glen Little as Blackwell tried to conjure up an equaliser.
And it nearly arrived on 64 minutes when Little's free kick picked out on-loan Arsenal youngster Bartley but his header sailed over the crossbar.
The 18-year-old then beat the offside trap but Cresswell was unable to convert his cross, while Hayes was denied Scunthorpe's second as Simonsen tipped his low drive round the post on a rare counter-attack by the visitors.
Credit to the Blades, they refused to panic and Quinn picked out Camara with a slide-rule pass but the striker was hustled out by Iron defenders as he closed in on goal.
Former Blade Forte came on with 16 minutes remaining and had a dramatic introduction. He had calls for a penalty waved away at one end, then entered the referee's notebook for the first booking of the match after dragging back Little.
Substitute Camara was then replaced himself after just 25 minutes of action, Lee Williamson taking his place for the final 12 minutes. Afterwards, Blackwell revealed Camara had played his final game for the club after a groin injury flared up again and has ruled him out for the last few weeks of the season.
Henderson headed over at the far post, Bartley failed to get any leverage on a header from Little's corner, while at the other end Nyron Nosworthy thwarted a three-against-one counter attack as he forced Martyn Woolford wide.
As five minutes of stoppage time was announced, the inspired Murphy did well to get down and clutch Henderson's header although the Blades striker was penalised for being offside.
But the Scunthorpe goalkeeper saved the best until last as he thwarted a close-range header from Bartley as he glanced goalwards a Little cross.
Chants of 'Blackwell out' echoed from a half-empty Kop and the chorus of boos which greeted the final whistle was deafening for a the defeated Blackwell.
Hero: Paul Hayes
Scunthorpe United arrived at Bramall Lane without a win at the ground in 52 years and in the absence of any heroes from the Sheffield team, Hayes deserves the accolade for his winning goal.
Villain: Joe Murphy
On a day when Sheffield United really needed a win to keep alive their play-off hopes, Scunthorpe goalkeeper Joe Murphy denied them with a string of fine saves. His display could ultimately mean the Blades missing out on a place in the top six.
45th minute: With the players and crowd ready for half-time, Donal McDermott crossed from the right and the unmarked Paul Hayes swept in the winner.
Russell Booth: Kept his card in his pocket until the last 15 minutes when Johnathan Forte became the first and only player to be booked in an easy game for the official.
While Kevin Blackwell's side may not be mathematically out of the play-off running, this defeat means it is now going to be very, very difficult for them to achieve a place in the top six of the Championship. These are tough times for Blackwell and for the Blades supporters.
Quote of the day
To be up near the top with seven games to go has been a phenomenal achievement by the staff.
– Kevin Blackwell asks for understanding from the supporters.
Championship: Barnsley home, Saturday, April 3.
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