Wigan Athletic 1 Port Vale 3
The 6-1 victory over Liverpool Under-21s in their opening game of the round-robin group stage last month had helped to kickstart Wigan's campaign.
And boss John Sheridan will be hoping this setback doesn't do the opposite in terms of their league form.
Latics - without senior figures such Joe Garner, Kal Naismith and Jamie Jones - started brightly but fell behind on 13 minutes.
Theo Robinson opened the scoring with a cool finish following a defensive misjudgement, and only a fine save from Owen Evans prevented David Worrall doubling the advantage.
A bad mistake from Darnell Johnson allowed Robinson to make it 2-0 on the half-hour mark, and it would have been 3-0 at the break had Cristian Montano's thunderbolt been a yard lower instead of hitting the bar.
Montano made amends 10 minutes into the second half when he strolled through some weak defending on the edge of the Latics box and slotted past Evans.
The home side pulled one back with 11 minutes to go through Tom Pearce's brilliant free-kick from 25 yards.
And there was almost a very nervy finale when first Charlie Jolley and then Olly Crankshaw were denied only by desperate Vale defending.