The 41-year-old guided Cardiff to the Premier League for the first time in their history last season, leading the side to the Championship title.
Cardiff have made a solid start in their fight to remain in the top flight, with the club gaining 17 points from 18 fixtures and currently sitting 16th in the table.
But Mackay's relationship with owner Vincent Tan has come under scrutiny in the past month, with the Malaysian reportedly issuing an ultimatum to the Scot to resign or be sacked.
It looked like Mackay would enjoy a stay of execution with the two set to meet to reconcile their differences.
Cardiff, however, have now decided to remove Mackay from his position a day after the 3-0 home defeat against Southampton, a game that saw protests against Tan from angry supporters.
A statement on the club's official website read: "The board of directors at Cardiff City Football Club have today relieved Malky Mackay of his duties.
"A new first team manager will be appointed and announced in due course."
Mackay's dismissal had a certain air of inevitability, despite chairman Mehmet Dalman saying the former Watford manager would be in charge for the foreseeable future last week.
Tan has proved a controversial figure at the Welsh outfit, not least for his decision to change the club's colours from blue to red at the start of last season.
