The club made the announcement on their official Twitter feed, adding that a search has begun for a new chief executive officer.
Club owner Katharina Liebherr, who in a reshuffle becomes non-executive chairman, said: "With great regret we have accepted the resignation of Mr Cortese.
"He has done a wonderful job and we very much wanted him to stay. A search has now begun for a successor.
"It is business as usual and we will ensure that the manager, team and staff at the club have all the help and support they need."
It is understood Cortese had been contemplating quitting due to divisions between him and Liebherr, who took over as the south-coast outfit's owner after the death of her father Markus Liebherr in 2010.
Cortese became executive chairman in 2009 after being instrumental in the Liebherr purchase of the club earlier that year, and has since overseen Southampton's rise from League One to the Barclays Premier League.
The Italian's departure is sure to cast doubt over the future of Southampton manager Mauricio Pochettino.
There was talk of Cortese considering his position in May, and Pochettino subsequently warned he too would leave Southampton if the executive chairman chose to do so.