Pablo Counago gave Blues the lead but the FA Cup Semi-Finalists took the points, inspired by a brace from Brian Howard.
Counago finished of a slick move on 20 minutes, steering in his 11th goal of the season after Velice Sumulikoski's shot had been parried by Luke Steele.
Howard levelled the scores seven minutes later with a neat finish before former Town loanee Jon Macken headed in early in the second-half.
Howard made it three from the penalty spot seven minutes from time after Blues had a spot-kick appeal turned down seconds later and then claimed his hat-trick with his corner going into the net, though the ball appeared to go in off David Wright.
Alan Lee and Counago both hit the woodwork and Blues matched the Wembley bound opponents until that late flurry brought a misleading scoreline.
The game survived a pitch inspection two hours before kick-off but Danny Haynes didn't survive the warm-up, Liam Trotter replacing him on the bench after the teenager pulled out with an injury.
Jon Walters was forced to stay behind at the Club for treatment for an on going knee problem with Jim Magilton making three changes to the side that drew with Sheffield United. Alex Bruce coming in for Dan Harding, Owen Garvan replacing Haynes and Alan Quinn in for Walters.
Howard sent the first opening over the bar, Counago repeating the feat at the other end before the Tykes' FA Cup Quarter-Final hero, Kayode Odejayi missing a gilt edged chance on the quarter hour, lifting the ball over the bar from eight yards.
It was Town who got the breakthrough five minutes later, a slick move involving Alan Lee and Quinn set up Sumulikoski, whose shot was saved by Steele but Counago converted the rebound for his first goal in 16 games.
Town were lifted by the goal and David Norris was robbed by Bobby Hassell as he prepared to shoot, then Counago linked up well with Sumulikoski, who showed a great touch before firing into the sidenetting.
Barnsley levelled on 27 minutes, skipper Howard taking advantage of Jamal Campbell-Ryce's pass to steer the ball past Stephen Bywater.
Diego Leon then tested Bywater from distance before Hassell picked up the first booking of the game.
Jason De Vos came to Town's rescue just before the break after Bywater's throw had gone straight to Howard before referee Darren Drysdale brought the first-half to an end.
Blues could have been ahead in the first minute of the second-half, Counago heading over from Garvan's free-kick but it was the home side who gained the advantage, Macken left alone to head in from three yards.
Town responded in style, Steele doing well to keep out Lee's goalbound strike, the Republic of Ireland international then hitting the woodwork with a powerful strike after Counago had released him.
The Spaniard then showed a great touch to wriggle free from his marker but Steele diverted his shot wide.
Counago then hit the bar with another strike after twisting inside the Tykes' box before Barnsley made the first change midway through the half, Sam Togwell on for Leon.
Tykes' boss Simon Davey made a second change for the last 15 minutes, Istvan Ferenczi replacing Odejayi.
Counago then went down in the box under a challenge from Nyatanga in the box but referee Drysdale turned away the appeals but seconds later pointed to the spot at the other end after Campbell-Ryce was judged to have been brought down by Sumulikoksi. Howard did the rest from the spot.
Blues then introduced Jordan Rhodes against the club he supported as a boy, Dan Harding joining him in a double change with Sumulikoski and Bruce making way for the final exchanges.
The Tykes' fourth came in bizarre fashion, Howard swinging in a corner and the ball appering to go in off Wright's head although the Barnsley skipper claimed his hat-trick
Trotter was given a run out for the last few seconds, coming on for Quinn and Lee went close to a consolation with virtually the last kick of the game.
The defeat sees Town drop to ninth, but still only three points off the Play-Off places.
Barnsley: Steele, Foster, Nyatanga, Souza, Kozluk, Campbell-Ryce, Hassell, Howard (Butterfield 90), Leon (Togwell 68), Macken, Odejayi (Ferenczi 75). Subs not used: Nardiello, Coulson. Booked: Hassell.
Town: Bywater, Bruce (Harding 84), De Vos, Naylor, Wright, Norris, Garvan, Sumulikoski (Rhodes 84), Quinn (Trotter 88), Counago, Lee. Subs not used: Colgan, Miller.
Att: 11,333
