And the decision was made last night to hand the reigns to assistant manager Gavin Skelton and Jim Thomson for the rest of the campaign.
The Championship side currently sit seventh in the table — 19 points behind Raith in the final play-off spot.
Fowler guided Queens to the play-offs last season, where they were knocked out by Rangers.
But a disappointing end to the campaign for the Doonhamers this year has seen any hopes of repeating that achievement slip away.
Chairman Billy Hewitson said: “It’s always disappointing when a manager leaves, we thank James for his efforts and wish him all the best for the future.”
Queens host Livingston at Palmerston on Saturday before travelling to promotion hopefuls Hibernian on the final day.
Queens sack manager Fowler
QUEEN of the South have parted company with manager James Fowler — with just two league games remaining.
Chewin' the Fat star is coming to Dumfries
Actress and comedian Karen Dunbar spoke to the Dumfries Courier ahead of her show at the Easterbrook Hall on Thursday night.
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