Newcastle's second-string fell behind in the early stages at the Saints' Staplewood Training Ground following Dom Ballard's strike before equalising in quick fashion courtesy of Garang Kuol, the Australia international's first goal for the club at under-21 level.
Southampton were reduced to ten men towards the half-hour mark following Derrick Abu's straight red card following a last-man challenge on Johnny Emerson but managed to retake the lead before the half-time whistle following Moses Sesay's strike.
Sam Amo-Ameyaw doubled the Saints' advantage in the second-half and despite Josh Donaldson halving the deficit for the visitors in the closing stages, Princewill Ehibhatiomhan's late effort regained Southampton's two-goal lead in the final minutes of the league encounter.
Newcastle's second-string, playing their last competitive fixture a month ago, fell behind in the sixth minute through Ballard as the Saints striker, who scored a hat-trick in last season's encounter with the young Magpies, fired the ball home from Sesay's pass.
United's youngsters, however, levelled the clash in the 13th minute through Kuol as the Australia international, who represented his country at the 2022 World Cup finals in Qatar, drilled a low effort beyond Southampton goalkeeper Gavin Bazunu.
Southampton were reduced to ten men towards the half-hour mark as Abu received a red card as Magpies winger Johnny Emerson, darting through on goal, was brought down on the edge of the box by the Saints right-back.
Despite their advantage, Newcastle fell behind for a second time as Sesay, a late inclusion to the starting XI after Welington's withdrawal, turned from provider to goalscorer in the closing stages of the first-half.
Newcastle were dealt a further blow in the 79th minute as Amo-Ameyaw doubled Southampton's lead before Donaldson, who signed his first professional contract with his boyhood club last summer, halved the deficit in the 86th minute after the Gateshead-born striker turned the ball home from Darren Palmer's cross.
However, in the 90th minute, Newcastle's hopes of salvaging a positive result were distinguished as Ehibhatiomhan's strike confirmed maximum points for the Saints to, ultimately, round off a disappointing afternoon on the South coast for Diarmuid O'Carroll's side.
Despite the defeat, Newcastle's youngsters currently remain in sixth place of the Premier League 2 and will be aiming to bounce back in positive fashion when hosting their Everton counterparts at Whitley Park on Friday, 24th January (kick-off 7pm GMT) in their next league game.
