The Championship club made their decision following the 3-0 home defeat to Birmingham City on Saturday.
"Barnsley Football Club can confirm first-team manager David Flitcroft has been relieved of his position at the football club," reads a statement on the Yorkshire outfit's official website.
The statement added that Barnsley will make no announcement on who their next manager will be until next week.
"There will be no further statements from the club until Wednesday December 4, 2013," it continued.
"The club would like to place on record their appreciation and thanks for what David, Martin and Guy have achieved."
Barnsley are currently at the foot of the table with 11 points from 17 matches.