Paul Harsley's youngsters claim vital three points away at Westleigh Park in the group stage of the Premier League Cup.
In a game that looked for all the world like it was heading for a 0-0 stalemate, up popped Jacob Brown in the 89th minute to head home the Reds winner.
Harsley sent out a more youthful looking side than in previous games, with Jack Walton returning between the sticks and Louis Wardle continuing on the right side of midfield; Jared Bird captained the Reds alongside teenage sensation, Matty Wolfe.
And it was Wardle who was first to test the home 'keeper, his shot though was comfortably held and the Reds denied an early lead.
Soon afterwards, Victor Adeboyejo, fresh from his appearance on the first team bench last weekend, he powered an effort towards goal from close-range which the Pompey 'keeper, Alex Bass parried away to safety.
Theo Widdrington looked the liveliest Pompey player in the first half and he twice blazed over from outside the box after the Reds defence struggled to clear their lines.
Before the break, Walton had to be alert to deny Leon Maloney who had found his way through to goal.
HALF TIME: Portsmouth 0-0 Barnsley
Almost from the restart, Romal Palmer tried his luck outside the box but his effort was straight at Bass.
The hosts had their best opportunity when Dan Smith countered from a Barnsley corner only to be denied once more by the alert Walton.
The game looked certain to end goalless, but just as we approached full time, Brown met Ben Williams' corner with power, heading beyond the helpless Bass and sending the Reds back up north with a priceless three points.
FULL TIME: Portsmouth 0-1 Barnsley
Reds: Walton (GK); Smith, Fielding, Lund, Williams; Wardle, Wolfe, Bird (Dolan '78), Palmer; Adeboyejo, Brown.
Unused: Tingle, Swinburn, Miller, Ocran.