Jordy Clasie and Ryan Bertrand scored as the Saints sealed a semi-final berth at the Emirates.
Arsenal failed to progress beyond the EFL Cup quarter-finals for the sixth season in a row as Southampton beat them to a semi-final spot with a 2-0 win at Emirates Stadium.
First-half goals from Jordy Clasie and Ryan Bertrand did the damage for Southampton, who had lost 2-1 on this ground earlier this season in the Premier League.
The League Cup remains the only domestic honour Arsene Wenger has never won with Arsenal, but there was little sign of an appetite to change that on Wednesday.
Wenger made 10 changes to his side and even the Frenchman's attire - tracksuit bottoms poking under his traditional long coat, rather than a suit - suggested this was not a highlight in the Gunners' schedule.
A first defeat since the opening day of the Premier League season, nevertheless, angered the home fans, who met the half-time and full-time whistles with widespread disapproval.
The Saints, by contrast, will be jubilant, having made their first semi-final in this competition in 30 years as Claude Puel continues to keep Southampton in impressive fettle.
Though he too made changes, Puel's side retained considerable steel, with the now routinely excellent Virgil van Dijk marrying physical strength and a calm head to keep a hesitant Gunners attack at bay.
A tense opening to the game was blown apart by Clasie when Sofiane Boufal's shot was blocked into his path and the former Feyenoord man blasted the Saints into the lead from 18 yards.
Arsenal pressed and probed in response, but their much-changed team lacked incision and Southampton defender Maya Yoshida went closest to levelling the scores when he perilously flicked Mohamed Elneny's free-kick just wide of his own goal.
The visitors had seen earlier counter-attacks fizzle out, but they sliced through the makeshift Gunners side to extend their lead before the break.
Boufal darted in from the left flank before feeding Bertrand, who took a touch before threading his shot through the crowd and past Arsenal's third-choice goalkeeper Emiliano Martinez.
Elneny - Arsenal's only survivor from Sunday's win over Bournemouth - was forced off before the break and replaced by Granit Xhaka, whose fizzing free-kick forced Fraser Forster into action early in the second half after the England man had also repelled Aaron Ramsey from close range.
That burst early in the period was weathered by Southampton, with Boufal and Sam McQueen seeing shots deflected wide as the Saints continued to make the game's better chances.
Wenger threw on Alex-Oxlade Chamberlain, scorer of three goals in the two previous EFL Cup rounds, but the former Saints man blazed over when well placed, sucking momentum out of the hosts' fightback as they meandered to elimination.