The group of cows made their way onto the carriageway between junction 34 and 35 of the M1.
National Highways said it had set speed limits and lane closures on the smart motorway area to warn traffic in the area.
The agency said: "Our Traffic Officers and @syptweet are en-route to the #M1 northbound between J34 (#Sheffield) and J35 due to a number of cows which have made their way onto the carriageway. We've set speeds and lane closures to warn approaching traffic.
"The cows have now made their way onto the J35 (#Rotherham) northbound exit slip road, approaching the roundabout. Our Traffic officers and Police are still en-route and are expected to arrive asap."
A short time later it added: "Traffic has now been STOPPED on the #M1 northbound in #SouthYorkshire between J34 (#Sheffield) and J25 (#Rotherham) to allow Police to round up the remainder of the cows which are still on the nearside verge. Delays are building on approach.
The traffic was released a short time later, at around 7am, and although the cows had been removed from the carriageway, there will still tailbacks of around two miles.