Lemina moves to the Premier League for an initial €17 million ($34 million AUD) that could rise to €20m ($40m AUD) if certain stipulations are met.
The 23-year-old joined Juve from Marseille in 2015, but was restricted to 15 Serie A starts and he will now look to kick start his career courtesy of a five-year contract at St Mary's.
Lemina made 42 appearances in all competitions for Massimiliano Allegri's side, scoring three times.
"I am really happy to have signed for Southampton," he said.
"I think that it was the project that attracted me the most and the one my family was really behind. I am really happy to be here."
Les Reed, Southampton's vice-chairman of football, said: "Mario is an outstanding talent who has already achieved a great deal in his career, winning a number of titles and gaining experience of playing on some of the biggest stages in world football.
"Investing in a player of Mario's quality and buying from a club like Juventus is a significant statement of intent from the club, and this is a purchase that has set another club-record fee.
"Mario still has his best years ahead of him, and we believe that he will not only be an excellent acquisition for us in the immediate future, but that he will also be able to realise even more of his potential here in the years to come.
"In a volatile market with some extraordinary and inexplicable transfers taking place, we are determined to go about our work in a diligent and correct way. This is a player who has won trophies and played in the Champions League final less than three months ago and he has signed for Saints amid a host of suitors.
"Mario will complement what is already an extremely strong group of midfield players in our squad, and we are excited about what he will bring to the group."
Lemina is Southampton's second recruit under new boss Mauricio Pellegrino after Poland Under-21 defender Jan Bednarek arrived from Lech Poznan on a five-year deal at the start of last month.
Saints begin their 2017-18 Premier League campaign at home to Swansea City on Saturday but were rocked this week by captain Virgil van Dijk handing in a transfer request.