McCall was the second-longest-serving manager in Scottish league football, behind Forfar Athletic's Dick Campbell, having taken charge at Fir Park in December 2010.
The former Everton and Rangers midfielder guided Motherwell to top-three finishes in each of his full seasons in charge - the club finishing as runners-up to Celtic for the last two campaigns.
However, Motherwell have made a dismal start to the 2014-15 campaign and sit second-bottom of the top flight with seven points from 12 matches.
A 2-1 defeat at St Johnstone on Friday represented a fifth straight league defeat and ninth loss of the season for McCall's men.
The 50-year-old has now chosen to step down, despite Motherwell indicating they were willing to keep faith with their manager.
"Motherwell FC can announce Stuart McCall has resigned from his position as manager," read a statement from the club on Sunday.
Derek Weir, speaking on behalf of the Motherwell board, said: "Everyone at Motherwell FC would like to sincerely thank Stuart for his contribution to the club, which will go down as one of the most successful managerial tenures in our 128-year history.
"We have been acutely aware of results this season, but everyone at Fir Park was confident in his, and the team's, ability to turn things around.
"However, following a long conversation this afternoon, Stuart felt it better for all concerned if he stepped down with immediate effect and we respect that.
"Assistant manager Kenny Black will take charge of first-team team affairs in a caretaker role to allow the board to start the process of recruiting a new manager, which will begin right away."
