Emergency services were called to Whispers nightclub in Barnsley town centre after reports a 30-year-old man had been stabbed at the venue.
Police and paramedics attended the scene at around 1.15am on Sunday and the injured man was taken to hospital shortly afterwards.
The man is said to have attended a private function inside the premises.
A South Yorkshire Police spokesman said: “Police are appealing for witnesses following an incident at Whispers on Regent Street in Barnsley at 1.15am today.
“A 30 year old man, who is believed to have attended a private function at the nightclub, received a stab injury and is now in a serious condition in hospital. Enquiries are now underway.”
Anyone with information is asked to call 101 quoting incident 64 of 18/12/16.