The Saints, who drew with Manchester United in the Premier League on Saturday, are 18th in the table.
First-team assistant coach Kelvin Davis will take charge of the team for the game against Tottenham on Wednesday.
Hughes, who also had a spell at the south coast club during his playing career, joined Southampton in March, two months after being sacked by Stoke City.
The club were one point above the relegation zone at the time and the 55-year-old led them to safety last season by securing two wins from their last four games.
However, they have struggled again this season and, across his spell, have won just three of their 22 league games.
Before the draw against Manchester United, Southampton were beaten 3-2 by relegation rivals Fulham and knocked out of the EFL Cup on penalties by Leicester.
Along with Hughes, assistant first-team manager Mark Bowen and coach Eddie Niedzwiecki have also left the club.
Southampton’s hierarchy were never going to make the same mistake as last season when they almost left it too late to sack Mauricio Pellegrino as they dropped towards relegation.
It meant added pressure on Mark Hughes, who presided over their survival, and his dismal win record of three wins from 22 league games made this decision inevitable.
Hughes has told regular hard luck stories this season but eventually the script becomes tired when performances, even though they were decent enough on occasions, simply do not get results.
Hindsight is a wonderful thing but Southampton may now reflect on their decision to give Hughes a three-year contract in the afterglow of retaining their Premier League status rather than thanking him for his efforts and returning to the drawing board.
Saints’ vice-chairman Les Reed and technical director Martin Hunter have already gone and the sweep has continued with Hughes’ sacking. These are changing times at St Mary’s.
As for Hughes, it is hard to see where he will go next after successive sackings at Stoke and Southampton in so short a time.
He now joins other old managerial heads like Sam Allardyce, David Moyes and Alan Pardew on the margins.