A goal in each half did the damage as Rovers Under-23s suffered a first defeat in 10 games away at Southampton in the Premier League Cup.
Rovers had the majority of the play at Staplewood Training Ground down in Hampshire, but two goals at crucial times proved critical for Damien Johnson's development squad.
Rovers went behind to Yan Valery's strike on the stroke of half-time, with Southampton doubling their lead just after the break through the experienced Matt Targett.
The visitors tried to rally and tested Jack Rose on numerous occasions and also hit the bar, but it simply wasn't to be.
Johnson handed a start to Lewis Travis, who had travelled down to the south coast straight after the first team's 3-3 draw against Crewe Alexandra in the FA Cup on Sunday.
Joe Grayson came into the XI for Rovers in one of two changes to the starting line-up; the other being Charlie Albinson, who deputised for Andy Fisher in goal.
Hosts Southampton included both Targett and Jake Hesketh in their XI - both of whom had played Premier League football for the club.
Saints went close to breaking the deadlock on 18 minutes when Nathan Tella tried his luck from the edge of the area, but his left-foot strike bounced back off the post.
Moments later, Rovers were dealt a blow when Callum Wright picked up an injury and had to be replaced by Connor Thomson.
In a tight first half, Lewis Mansell then played in Dan Butterworth, who fired narrowly over the upright on 24 minutes.
Rovers continued to press for the opener, with Travis producing an excellent run and cross, but nobody was lurking in the centre for a tap-in.
Just a couple of moments later and Mansell saw his shot on the turn saved by Saints stopper Rose.
And, just when it looked like both sides would go in at the break all square, the hosts took an unlikely lead.
Full-back Valery found himself in the box and he dug out a shot which beat Albinson at his near post after Rovers failed to clear their lines.
It was a cruel blow for Johnson's side going into the break in this top-of-the-table clash.
Just nine minutes into the second half and Saints scored another goal, as Targett expertly curled into the corner from a free-kick, past Albinson's despairing dive.
Rovers looked to respond and another good Travis cross caused problems for the Saints defence, but Valery defended well to stop Matty Platt from finding the net just after the hour mark.
Time was running out for Rovers to find a way back into it and Butterworth tried his luck from range with 15 minutes remaining, but it was straight at Rose.
With five minutes to go, Willem Tomlinson blasted wide as the visitors upped the tempo in a bid to salvage something from the game.
Sam Hart headed against the bar in the closing stages and Mansell saw a volley blocked by Alfie Jones on the follow-up, as Rovers tasted defeat for the first time in 10 games, with Saints replacing Rovers at the summit of the PL Cup table.
SOUTHAMPTON: Rose, Valery, Cook, Jones (c), Targett, O'Connor, Hesketh, Teller, Slattery, Afolabi (Mdlalose 88), Barnes.
Subs not used:Latham, Little, Wood, Smallbone.
Goals: Valery (45+1), Targett (54).
Booked: Targett, Slattery, Hesketh.
ROVERS: Albinson, Travis, C. Doyle (Paton 73), Platt, Grayson, Hart, Hardcastle (c), Tomlinson, Wright (Thomson 20), Butterworth, Mansell.
Subs not used: Dillon, Thompson, Buckley.