The Austrian departs the Saints alongside assistant Richard Kitzbichler after Sunday's 4-1 defeat to Newcastle, which has left them in the Premier League relegation zone.
A Southampton club statement read: "Hasenhuttl departs having made a significant contribution to the club, overseeing some memorable results and also playing a key role in the development of our club infrastructure, identity and playing squad.
"However, we now believe it is an appropriate time to make a change.
"Everyone involved with the club would like to express their sincere thanks to Ralph for all of his efforts, as well as the unwavering commitment he has shown throughout his time as manager."
First-team coach Ruben Selles will take charge for Southampton's Carabao Cup third round tie against League One side Sheffield Wednesday.
Hasenhuttl was appointed in December 2018 and led the club to an 11th-placed finish in his first full season, before two consecutive 15th-placed finishes.
But there were some sobering results along the way including two 9-0 thrashings at the hands of Leicester and Manchester United.
The Saints have started the current campaign in dismal fashion and sit 18th in the table having one once in their last nine games.
Hasenhuttl, who was the division's fourth-longest serving manager, has become the fifth to have been dismissed in the Premier League so far this season.
Scott Parker, Bruno Lage, Thomas Tuchel and Steven Gerrard were sacked by Bournemouth, Wolves, Chelsea and Aston Villa respectively.
