A first-half goal from Marcus Barnes proved enough to see off a young Foxes side that were looking for their seventh win in eight games in all competitions.
City had back in September at their Staplewood training ground, when goals from Layton Ndukwu and Chris Dusabe handed them victory on that day.
Since then, Leicester have gone on to register eight wins in 11 games in all competitions, most recently advancing to the FA Youth Cup Fourth Round with a at King Power Stadium on Wednesday.
While Southampton sat 11th going into the clash, City were second in the table and were looking to close the gap on league-leaders Chelsea, who had played two games more than the Foxes before the tie.
City started well and could have gone ahead in the first minute. Harvey Barnes’ shot was blocked and the rebound fell to captain Ben Chilwell, who slotted just wide.
Admiral Muskwe tried one from range on the quarter-hour mark as his shot was tipped behind by goalkeeper Harry Isted.
Southampton took the lead on 18 minutes. Josh Sims, , burst down the wing and set up Barnes, whose shot sneaked past Max Bramley in the Leicester net.
The visitors came close to doubling their on 39 minutes when Barnes took the ball round Bramley, but the shot-stopper recovered well and smothered the ball at the feet of the No.9.
A free kick two minutes before half-time from captain Armani Little then flew just past Bramley’s upright as the wind carried the ball towards goal – 1-0 to the Saints at the break.
Southampton threatened again on 54 minutes, but Bramley was wise to Barnes’ shot, and palmed the ball to safety.
Fox had his attempt saved by Isted just after the hour mark while at the other end, Elliott Moore was on hand to block substitute Callum Slattery’s effort.
A late scramble in the opposition box first saw Ndukwu’s shot saved before Moore thought he had found an equaliser with a close-range header, only for his goal to be disallowed with seven minutes to go.
Muskwe then latched onto a through-ball and hit a sweet half-volley that crashed off the Southampton crossbar – City coming agonisingly close to an equaliser, but couldn’t find one.
The young Foxes continue their 2014/15 league campaign in the New Year when they host West Bromwich Albion at Belvoir Drive on Saturday 10 January.
CITY: Bramley, Knight (King 72), Chilwell (c), Choudhury, Moore, Kipre, Ndukwu, Miles, Muskwe, Barnes, Fox
SUBS NOT USED: Johnson, Yates, Fura, Mitchell
SOUTHAMPTON: Isted, Mason, Debayo, Jones, Clinton, Cook, Demkiv (Slattery 58), Little (c), Barnes (Olomola 72), Willard (Lea 83), Sims
SUBS NOT USED: Cull, Bakary
GOAL: Barnes 18
REFEREE: Mr G Hubbard