Rovers' five game unbeaten run came to an end as Southampton ran in three second half goals at the St Mary's Stadium.
Tranmere had been the better side in a goalless first half, but once Southampton took the lead early in the second period the home side took control of the game and ran out comfortable winners.
Rovers were unchanged following the win over Aldershot in the FA Cup, with Craig Curran, Ian Thomas-Moore and Terry Gornell once again making up the front three.
A scrappy start to the contest saw both sides give up possession easily and the final ball failing to create any chances. Ian Goodison did well to prevent David Connolly from having a clear shooting opportunity on 11 minutes while a John Welsh cross went out of play as Rovers quickly counter attacked at the other end.
Ian Thomas-Moore volleyed over the bar from Shaleum Logan's deep cross on 12 minutes and another Logan cross had to be cleared by Chris Perry five minutes later.
Craig Curran fired over on 19 minutes while Marlon Broomes cleared Lloyd James' free kick soon after.
Possession was being shared fairly evenly between the two sides but it was Rovers who were looking the more creative going forward. A long range Welsh effort went wide of Bartosz Bialkowski's goal on 26 minutes and the keeper saved another shot from the midfielder five minutes later.
Bookings for Ian Goodison and Rickie Lambert on 36 minutes brought a loud reaction from the home fans and led to a spell of stoppages in the game for niggly challenges.
Morgan Schneiderlin's shot was parried by Luke Daniels on 37 minutes with the keeper gathering at the second attempt just in front of the rapidly approaching Lambert.
The final chance of the first half came deep in injury time with Welsh sending another well struck effort just wide of Bialkowski's right hand post.
A foul on Welsh 25 yards out gave Rovers a dangerously positioned free kick four minutes into the second half but Gareth Edds' curled attempt went over the bar.
The home side took the lead with their first attack of the second half. Southampton passed the ball around the edge of the Rovers area before it found Dan Harding who shot powerfully into the net with his left foot from 12 yards.
On 64 minutes the Saints were awarded a penalty when the ball struck the arm of Broomes from a corner. Spot kick duties fell to former Bristol Rovers striker Lambert who doubled his side's lead with an emphatic strike.
A double change saw Michael Ricketts and Chris Shuker replace Gornell and Edds with just over 20 minutes remaining, but soon after Southampton went 3-0 up. This time a foul by Curran on Harding gave Alan Pardew's side a free kick 30 yards out and Lambert brilliantly sent his shot over the wall and past Daniels.
A final change brought the introduction of Paul McLaren in place of Welsh, while the home side were almost given a fourth when Daniels dropped a cross into the path of Lambert only to quickly recover and block his shot.
Southampton: Bialkowski, James, Perry, Jaidi, Harding, Hammond, Schneiderlin (Wotton 65), Lallana, Antonio, Lambert (Mellis 84), Connolly (Papa Waigo 84). Substitutes not used: Forecast, Thomas, Trotman, Mills.
Tranmere: Daniels, Logan, Broomes, Goodison, Bakayogo, Welsh (McLaren 77), Taylor, Edds (Shuker 69), Thomas-Moore, Gornell (Ricketts 69), Curran. Substitutes not used: Collister, Cresswell, Barnett, Fraughan.
Referee: Danny McDermid
Yellow cards: Goodison (36), Lambert (36), Schneiderlin (49), Broomes (64)
Away attendance: 350