Five unbeaten going into the clash at the Lamex Stadium - broadcast live by Spurs TV - we were undone by a goal in each half.
Southampton's somewhat fortuitous opener arrived as a real blow in the final seconds of the first period as Matt Targett's free-kick flashed through a crowd of players in the penalty area before nestling in the far corner.
Shaq Coulthirst went close twice before Saints struck the decisive blow through Josh Sims on the break in the 72nd minute.
Ugo Ehiogu fielded Andros Townsend, Harry Winks, Coulthirst and Milos Veljkovic as the team looked to make it three wins on the spin.
It was no surprise to see Townsend fire in the first effort of note, a trademark turn in off the right and shimmy before fizzing inches over on nine minutes.
Southampton replied within seconds as Sam McQueen broke into the box from the right, chopped onto his left foot and took aim, but Tom Glover in goal was alert and palmed away to his right.
Armani Little's in-swinging corner caused alarm shortly afterwards as first Cameron Carter-Vickers then Anton Walkes cleared in front of goal.
Coulthirst's low drive didn't seriously trouble Saints goalkeeper Paulo Gazzaniga midway through the half and the next chance followed on the half-hour when Carter-Vickers blocked Jake Hesketh's shot and the ball ballooned up for Ryan Seager to try his luck with an overhead kick, but his effort rattled the crossbar.
Coulthirst could only divert Anthony Georgiou's rasping cross from the left wide and it looked like the teams would go in level at half-time until the visitors grasped the lead in the final seconds of the half.
Targett lined-up his free-kick from the right and whipped in towards the six-yard box. The ball evaded everyone and left Glover helpless as it skidded home.
We were back on the front foot at the start of the second half.
Coulthirst rapped a shot into the side-netting after initially getting in the way of Townsend's effort and the pair combined again on 48 minutes when Coulthirst swept the England international's cross just wide.
Coulthirst went close again on 58 minutes. Picked out by Winks right of centre in the box, the striker showed quick feet to lose Targett but rifled across goal and just wide.
Townsend and Winks were replaced by Emmanuel Sonupe and Japhet Tanganga on 63 minutes, Tanganga's league debut for the Under-21s. Luke Amos switched from left-back to central midfield as part of the alteration.
Southampton were now set-up for the break and sprung us to go 2-0 up on 72 minutes. Hesketh played the ball in behind Tanganga for Sims who ran clear one-on-one before slotting past Glover.
Gazzaniga excelled himself in the 78th minute when he got down to divert away Carter-Vickers' snapshot. Georgiou latched onto the loose ball and fired low back across goal but there was no way through.
Substitute Will Miller couldn't get a clean connection after Sonupe left two defenders in his wake five minutes later with Georgiou's follow-up blocked this time by Ollie Cook.
Georgiou and Sonupe continued to work down both flanks but it was one of those nights in front of goal.
Spurs U21s: Glover, Walker-Peters, Amos, Lesniak (Miller 74), Carter-Vickers, Veljkovic (c), Townsend (Sonupe 63), Winks (Tanganga 63), Coulthirst, Walkes, Georgiou. Substitute (not used): Voss (GK).
Southampton U21s: Gazzaniga, Gape (c), Targett, Reed, Cook, Wood, McQueen (Johnson 74), Little, Seager, Hesketh (Slattery 80), Sims. Substitutes (not used): Jones, Debayo, Lewis (GK).