Paul Parry, who scored his first goal for the club, a Neil Mellor volley and a Jon Parkin strike ensured the visitors left south Yorkshire with all three points.
Iain Hume started alongside Kayode Odejayi as Barnsley boss Simon Davey rang the changes up front.
But it was Hume's inclusion that took many by surprise as he had been working his way back to full fitness after his recovery from a fractured skull.
For the visitors defender Youl Mawene failed to recover from a hamstring injury in time but Ross Wallace played.
It was Barnsley who started the brighter and Odejayi went close with an athletic header from the edge of the box.
Defender Bobby Hassell, who enjoyed a productive opening period for the hosts, managed to pick out the towering striker and a superb one-handed save from Andy Lonergan in the Preston goal was required to push the ball wide.
Veliche Shumulikoski had a goal-bound volley blocked before Parry put the visitors ahead against the run of play in the 10th minute.
Fellow front man Mellor played a well-timed through ball and Parry, who did well to cut in from the right flank and hold off the challenges of Luke Potter and Darren Moore, curled the ball beyond Tykes stopper Luke Steele and into the top-left corner of his goal.
Jacob Butterfield and Odejayi then went close in yet another dominant period for the hosts.
Tykes defender Moore then headed just over, but a second arrived for the Lilywhites in the 32nd minute when Mellor stole into the box to fire home.
A Shumulikoski free-kick was headed back into the box by Parry and, while a bemused Barnsley defence appealed for the offside, Mellor struck a sweet right-footed volley beyond Steele.
Barnsley came out fighting after the interval and Lonergan did well to push a Jamal Campbell-Ryce header to safety.
And minutes later defender Stephen Foster had a penalty claim waved away after he went down under the challenge of two Preston defenders.
A keen tussle between both side's midfields then ensued and Odejayi stretched desperately - but to no avail - for a cross from Potter which eventually went for a corner, while Moore glanced a Hume free-kick just wide as the home side looked to pull a goal back.
But the game was as good as over in the 72nd minute when former Reds youngster Parkin notched a third for Preston.
Billy Jones had pumped the ball into the box and Parkin did well to chest the ball down and fire a deflected shot beyond Steele to wrap up proceedings.