The 24-year-old, who is the club’s top scorer this season, will require surgery on a cruciate ligament injury and faces about six months on the sidelines.
That means Rodriguez, whose red hot form saw him handed an England start earlier this season, will not figure for Saints again this campaign and also forces him out of England boss Roy Hodgson’s plans for the World Cup in Brazil this summer.
A statement from Saints said: "Southampton Football Club can confirm that, following his injury on Saturday against Manchester City, Jay Rodriguez has been diagnosed as having suffered a rupture to his anterior cruciate ligament.
"This will keep him out of action for six months and regrettably means he will not be fit for selection by the English national team at this summer’s FIFA World Cup in Brazil."
Saints director Les Reed added: "Jay is naturally disappointed, but is determined to get back playing for Saints as soon as possible.
"Everything will be done to return him to full fitness and deliver a speedy recovery.
"Jay has asked us to pass on his gratitude to everybody who has shown him their concern and sympathy during the 48-or-so hours since his injury.
"Everyone at the Club now wishes Jay well and we will support him, along with his family, to ensure that an international career plays a big part in his future."