Saints went into the game in the knowledge that victory would lift them above their opponents and into second place in the Barclays U21 Premier League Elite Group.
But Martin Hunter's team struggled to get going at Moss Lane, and were beaten courtesy of Jesse Lingard's well-taken strike 20 seconds after half time.
There was plenty of endeavour and intent from a young Southampton side, but they struggled to find a way past a sturdy United defence.
Hunter made two changes from the side that beat Arsenal last Thursday, one of which saw Cody Cropper recalled between the sticks in place of Paulo Gazzaniga.
Jack Stephens also returned to the team having missed last Thursday's win over Arsenal with a knee injury to replace Matt Targett, who played for Scotland's Under-19s earlier in the day.
That meant Calum Chambers shifted to right-back, with Matt Young moved over to the left of the defence.
Included in the hosts' starting XI was Nick Powell, who has appeared for the United first team this season.
The game was stopped for a lengthy period after just six minutes when Chambers clashed heads with Marnick Vermijl in the Saints box. Both players were forced off a result, with Bevis Mugabi coming on to make his first Elite Group appearance at the heart of the visiting defence and Stephens moving to right-back.
The reshuffle seemed to benefit the home side, with Warren Joyce's side finding themselves in the ascendancy in the opening stages.
The first chance of the match went United's way, as some good play down the left saw the ball find Lingard but the midfielder poked his effort wide.
The hosts were close to opening the scoring on the 20-minute mark when Powell pushed the ball past Cropper and cut back for Lingard, but Stephens was on hand to intercept the pass in the nick of time.
At the other end, United right-back Frederic Veseli nervously headed the ball back towards his own goal but goalkeeper Ben Amos was quick off his line to claim it ahead of Jake Sinclair.
A chance fell to United on the half-hour when a corner from the right was half-cleared to Ryan Tunnicliffe, but he powered his close-range volley well over Cropper's goal.
There was a hairy moment for the Saints defence seven minutes before the break when Adnan Januzaj drifted past Young and flashed a low ball across the face of goal that Mugabi was forced to scramble behind.
HALF TIME: MANCHESTER UNITED U21S 0-0 SOUTHAMPTON U21S
After a dull opening half, the game sprang to life just 20 seconds into the second period.
From the kick-off, United played the ball down the left and substitute Reece James played in LINGARD to fire across Cropper and inside the far post for one-nil.
Saints went in search of a leveller and came close as Omar Rowe's low cross looked destined to be hooked in by Sinclair, but United defender Michele Fornasier got across to block.
The hosts almost made it two-nil ten minutes into the second half when a corner was flicked on by Fornasier to Tom Thorpe, but the United skipper was denied by Cropper.
Coach Hunter looked to liven his side up just after the hour mark, throwing on Gboly Ariyibi and Sam Gallagher in place of Omar Rowe and Lloyd Isgrove.
But United continued to have the better of the chances, and should really have sealed victory seven minutes from time when a low ball from the left found Powell a few yards out but he swept it inches over the bar.
Saints threw men forward for the final stages but their opponents held firm to record a one-nil win and strengthen their own position in the Elite Group.
FULL TIME: MANCHESTER UNITED U21S 1-0 SOUTHAMPTON U21S
Manchester United U21s: Amos, Veseli, Thorpe (c), Fornasier, Vermijl (James 10), Januzaj, Ekangamene, Tunnicliffe, Cole, Powell, Lingard. Subs (not used): Sutherland (GK), Blackett, Lawrence, van Velzen.
Goals: Lingard (46)
Southampton U21s: Cropper, Chambers (Mugabi 10), Stephens (c), Turnbull, Young, Gape, Reed, Moore, Isgrove (Ariyibi 62), Sinclair, Rowe (Gallagher 62). Subs (not used): Johns (GK), Curtis.
Referee: Steven Copeland