The 21-year-old, who made 32 appearances for Valladolid in all competitions in 2019-2020, has signed a four-year deal at St Mary's Stadium.
Salisu is Southampton's second signing of the close season following the arrival of Kyle Walker-Peters from Tottenham on Wednesday (AEST).
Manchester United, Atletico Madrid and Real Madrid were all linked with the highly rated defender, but he is pleased to have joined Ralph Hasenhuttl's side.
"It is a very good feeling to be a Southampton player. I am very happy to be part of the club and I am looking forward to starting here," Salisu said in a Southampton statement.
"Southampton is a club with a very rich history in developing young players, so it is a very good club for me to develop my skills and to learn a lot as a young player.
🗣 "I am very happy the fans are welcoming me so warmly and that they are happy with me."— Southampton FC (@SouthamptonFC) August 12, 2020
Get Mohammed Salisu's reaction to signing for #SaintsFC:
"I know that the team expects a lot from me and I'm going to try to give all I can to help the team."
Southampton finished 11th in the Premier League last season and are looking to rebuild, having also sold midfielder Pierre-Emile Hojbjerg to Tottenham for £20m.
Hasenhuttl is pleased with his latest capture and is expecting Salisu to slot straight into the side.
"This is an important signing for us, Mohammed is a player who fits our profile well," the Austrian said.
"He is young and has a great amount of potential, but he is also someone who has the qualities to come in and help the team as soon as he is up to speed with our way of playing.
"I like what I have seen of him so far in his games with Valladolid. He is strong defensively, is calm with the ball - something that is important in our team - and he has good speed too.
"I think he will fit in well with us and that this will be a good place for him to develop his game further. We have some good centre-backs here and this will be another strong option for us."