The Red Devils recovered at St Mary's to avoid crashing out of the cup to League One opponents for the second successive season.
Southampton threatened to repeat their famous 1976 Wembley win over United when Richard Chaplow gave them a deserved half-time lead against the much-changed Premier League leaders.
But, as in Tuesday night's dramatic comeback at Blackpool, the introduction of the veteran Ryan Giggs helped change the game, with goals from Michael Owen and Javier Hernandez putting the visitors into the last 16.
Having admitted making a mistake by fielding a weakened side in last season's shock third-round home defeat to Leeds, United boss Sir Alex Ferguson had gone strong against Liverpool earlier this month.
But he appeared to forget the lessons of the Leeds loss in picking his team on Saturday.
Buoyed by not having to face the likes of Wayne Rooney, Dimitar Berbatov and facing a makeshift defence and rookie goalkeeper, Southampton sensed an upset from the off.
The former Premier League stalwarts were almost given even greater cause for optimism in the eighth minute when the ball deflected through to Dan Harding, who coolly clipped home.
Unfortunately for him, the assistant referee's flag was already up.
But that did not deter the League One high-fliers, who almost profited from slack United defending when Rickie Lambert curled too close to debutant keeper Anders Lindegaard.
Liverpool-born striker Lambert also fired two free-kicks narrowly over, while captain home captain Jose Fonte failed to get proper contact on a corner which was pouched by Lindegaard.
United, meanwhile, were looking very much like a side which had rung the changes.
Making his first start for almost four months in a withdrawn role, Owen provided an early threat, steering Fabio's inside pass narrowly wide before clattering the far post with a mishit cross.
But that was as good as it got for the visitors as the in-demand Alex Chamberlain, reportedly a target for United, began to come more and more into the game.
The 17-year-old was involved in what should have been an opener a minute before the break when Guly nodded over Danny Butterfield's cross from six yards.
But Chaplow spared his team-mate's blushes moments before half-time when he broke into the United box and slammed the ball into the roof of the net at Lindegaard's near post.
If United fans were expecting the cavalry to arrive for the second half, they were initially disappointed as Wes Brown replaced Fabio in a defensive reshuffle.
With no sign of an equaliser in the opening 13 minutes, Ferguson sent on Giggs and Nani for Anderson and Darron Gibson, controversially ignoring Rooney and the on-fire Berbatov.
However, the double-substitution was quickly vindicated, Jonny Evans nodding Giggs' free-kick too close to Bartosz Bialkowski and Owen flicking Nani's low cross over the bar.
The equaliser soon followed in the 65th minute, with Gabriel Obertan's drilled cross deflecting right into the path of Owen, who nodded home his first goal since September.
Southampton came roaring back and marauding left-back Harding lashed a cross-cum-shot inches wide.
Saints sent on Ryan Dickson for Lee Barnard for the final 18 minutes before Nani fired a low half-volley straight at Bialkowski.
But the keeper was helpless as United completed their latest comeback 14 minutes from time, Fonte's slip allowing Javier Hernandez to receive Giggs' through ball and ghost an improvised finish into the net.
Southampton threw on Dany N'Guessan for Guly before Lindegaard flapped at a Harding cross and Chaplow left the field to a standing ovation, with Oscar Gobern coming on.
If the changes were meant to give Southampton a second wind, they failed to have the desired effect as United comfortably saw out time.