Crewe Alexandra ended their run of four straight defeats to deny League One play-off contenders Sheffield United the chance to go fifth in the table.
Harry Maguire and Jonathan Forte both placed chances wide for the Blades in the first half.
Arsenal loan man Chuks Aneke broke the deadlock on 49 minutes with a powerful volley from the edge of the area.
Joe Ironside went close to an equaliser for the visitors but his strike was well saved by Steve Phillips.
The Blades had already made sure of their play-off place but, with one game remaining, they now look likely to finish fifth or sixth and be at home in the first leg of their play-off semi-final.
After beating Southend at Wembley on 7 April, Johnstone's Paint Trophy winners Crewe's testing final six league matches of the season were all against teams in promotion contention.
VIEW FROM THE DRESSING ROOM
Alex boss Steve Davis told BBC Radio Stoke:
"We've had a bad spell since Wembley. I have found it difficult to pick them up after the final because Wembley is associated with the end of the season.
"We have had six really tough games after the final. But we had pride to play for and the players responded with a real battling performance which also had some quality too.
"We didn't deserve to lose at Yeovil on Saturday. I was really pleased with the way we performed there. But the boys can now take real confidence from this win because it shows that they can do it against the top teams.
"We won it with a great goal from Chuks, worthy of winning any game. He is capable of doing that. He was excellent."
Blades caretaker manager Chris Morgan told BBC Radio Sheffield:
"I wanted to look at a different setup away from home with the play-offs in mind. I also need to rest a few.
"We changed things up a little bit tonight - we had a look at some different players.
"The 11 we've had on the pitch at the start is well strong enough to win the game - we've tested the goalkeeper, we've had set-plays, and they have one chance and they win the game.
"Anyone that's seen us play this year will say that has probably been our Achilles heel. When we're on top and we're created chances we're not ruthless in front of goal."
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