The Blades suffered a first defeat in seven matches in front of Bramall Lane's biggest crowd of the season.
A total of 24,495 were inside the Lane but Sam Winnall's second half header decided this South Yorkshire derby.
There were no late heroics from Nigel Clough's side on this occasion as Danny Wilson marked his return to S2 with a victory.
Clough made four changes for the visit of Barnsley. McCarthy, Wallace, Campbell-Ryce and Baxter all returned to the starting line-up. McGahey, Reed and McNulty dropped to the bench, whilst Davies missed out.
Kicking towards the Kop in the first period, the early action was fast and furious, both teams won a corner inside the opening three minutes, whilst Murphy was actively putting Barnsley on the back foot. He had already supplied a cross which Scougall and Baxter almost got on the end of and then the Scottish winger showed too much pace for Cranie - from the result free-kick, Turnbull had to claw away a header from Alcock.
Expectedly, the frantic action abated, although chances were created at both ends. Winnall turned the ball over the crossbar after Dudgeon evaded Campbell-Ryce and Alcock to deliver a cross, whilst at the other end Baxter and Harris linked up at pace to present a chance for Wallace but Dudgeon managed to get a deflection on the shot. Soon after, on 20 minutes, Baxter's quick free-kick found Murphy but his shot lacked power.
As half-time approached the chances did begin to dry up, although Murphy and Barnsley's Berry tried their luck from distance and a good move between Campbell-Ryce and Baxter almost made a chance for Wallace.
Both managers resisted the urge to make changes at the interval and despite the first five minutes of the second half being end-to-end, goalscoring opportunities were not forthcoming.
A Harris sidefoot was blocked from Campbell-Ryce's cross before Barnsley grabbed the initiative on 56 minutes. Cole's shot was pushed around a post by Howard but from the resulting Bailey corner, WINNALL rose highest to head home from six yards. A mix-up between Campbell-Ryce and Alcock almost presented Winnall with another soon after.
Clough's response was to throw on McNulty, whilst long-range efforts from Harris and Murphy were off target. United were now forcing the issue with Baxter seeing a lot of the ball. On 72 minutes he provided the free-kick which saw an unmarked McNulty head over.
Persistent timewasting from goalkeeper Turnbull saw him booked on 78 minutes and Flynn was United's second change, replacing Campbell-Ryce. A Harris cross caused panic in the Barnsley box but Turnbull was off his line quickly to deny Scougall.
Porter was the last change but United's attacks were not producing chances, with the last ball going astray. Four added minutes was announced just as Flynn weakly shot at Turnbull. McCarthy was thrown forward as an auxiliary striker but the last chance to level was a Harris free-kick which Turnbull repelled.
Blades: Howard, Alcock, Harris, Wallace (McNulty 59), Doyle, Murphy, Scougall, Baxter (Porter 85), McEveley, Campbell-Ryce (Flynn 81), McCarthy.
Unused: Basham, McGahey, Reed, Turner.
Barnsley: Turnbull, Dudgeon, Berry, Nyatanga, Cranie, Hourihane, Winnall (Hemmings 84), Williams, Ramage, Bailey, Cole (Bree 90).
Unused: Treacy, Davies, M'voto, Trotta, Lita.