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."
Chuks Aneke has an effort at goal from just outside the box which goes wide of the right-hand post.
Chuks Aneke takes a shot from deep inside the box clearing the bar.
Substitution Jordan Chapell on for Barry Robson.
Joe Ironside has shot on goal from just outside the box which goes wide of the right-hand upright.
Byron Moore takes the inswinging corner, save by George Long.
Substitution Jonathan Forte goes off and Joe Ironside comes on.
Shot from just outside the area by Jonathan Forte goes over the bar.
Jamie Murphy fouled by Byron Moore, the ref awards a free kick. Free kick taken by Neill Collins.
Direct free kick taken by Harry Davis.
Booking The referee shows Neill Collins a yellow card for unsporting behaviour.
Handball decision goes against Neill Collins.
Barry Robson fouled by Kevin Stewart, the ref awards a free kick. Barry Robson has a direct shot on goal from the free kick, Steve Phillips makes a save.
48:52 Goal scored
Goal! - Chuks Aneke - Crewe 1 - 0 Sheff Utd Chuks Aneke finds the back of the net with a goal from long range to the top right corner of the goal. Crewe 1-0 Sheff Utd.
Kevin McDonald fouled by Ajay Leitch-Smith, the ref awards a free kick. Barry Robson takes the free kick.
Ajay Leitch-Smith challenges Darryl Westlake unfairly and gives away a free kick. Darryl Westlake restarts play with the free kick.
The referee blows his whistle to start the second half.
Substitution Jamie Murphy comes on in place of Danny Higginbotham.
45:00 +1:50 Half time
Half Time The half-time whistle blows.
Foul by Barry Robson on Byron Moore, free kick awarded. Strike on goal comes in from Harry Davis from the free kick.
Outswinging corner taken right-footed by Oliver Turton from the right by-line.
Shot by Chuks Aneke. George Long makes a save. Corner taken by Oliver Turton, Harry Maguire manages to make a clearance.
Chris Porter gives away a free kick for an unfair challenge on Chuks Aneke. Mark Ellis takes the free kick.
Corner taken by Barry Robson, Byron Moore makes a clearance.
Outswinging corner taken from the left by-line by Barry Robson, Mark Ellis makes a clearance.
Free kick awarded for an unfair challenge on Byron Moore by Darryl Westlake. Direct free kick taken by Harry Davis. Mark Ellis takes a shot. George Long makes a save.
Corner taken short by Byron Moore. A cross is delivered by Byron Moore, clearance made by Neill Collins.
Shot by Michael Doyle. Save by Steve Phillips.
Inswinging corner taken left-footed by Barry Robson from the right by-line to the near post, Mark Ellis makes a clearance. Corner taken left-footed by Barry Robson to the near post, Kelvin Mellor manages to make a clearance.
Mark Ellis gives away a free kick for an unfair challenge on Chris Porter. Free kick crossed left-footed by Barry Robson from left channel, clearance by Mark Ellis.
Oliver Turton takes a shot. Blocked by Neill Collins. Inswinging corner taken right-footed by Oliver Turton, Harry Maguire makes a clearance.
Effort from 20 yards by Callum McFadzean. Steve Phillips makes a save. Chris Porter takes a shot. Steve Phillips makes a comfortable save.
The referee blows for offside against Chris Porter. Steve Phillips restarts play with the free kick.
The referee blows for offside against Chris Porter. Free kick taken by Steve Phillips.
Jonathan Forte produces a left-footed shot from just outside the box that goes wide left of the goal.
The official flags Jonathan Forte offside. Indirect free kick taken by Steve Phillips.
Foul by Ajay Leitch-Smith on Harry Maguire, free kick awarded. George Long takes the free kick.
Shot by Bradden Inman from outside the box goes over the net.
Barry Robson challenges Bradden Inman unfairly and gives away a free kick. Direct free kick taken by Kevin Stewart.
The assistant referee signals for offside against Kevin McDonald. Free kick taken by Harry Davis.
Foul by Kevin McDonald on Kelvin Mellor, free kick awarded. Harry Davis takes the free kick.
Kelvin Mellor gives away a free kick for an unfair challenge on Kevin McDonald. The ball is sent over by Barry Robson, Michael Doyle takes a shot. Save made by Steve Phillips. Corner taken left-footed by Barry Robson to the near post, Byron Moore makes a clearance.
Shot from just outside the box by Jonathan Forte goes over the bar.
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