Former Hertha player and reserve team coach Pal Dardai will take over, assisted by Rainer Widmayer, the club said.
Hertha have lost five and won just one of their last seven league games and Wednesday's defeat dropped them to 17th in the table, two points above last-placed Borussia Dortmund.
"Hertha part ways with coach Jos Luhukay with immediate effect," the club said in a statement. "After analysing the current situation, the club decided on Thursday to also part ways with assistants Markus Gellhaus and Rob Reekers."
The 51-year-old Dutchman took over Hertha after they were relegated to the second division in 2012 and helped them win promotion in his first season in charge.
He is the fifth Bundesliga coach to lose his job this season.
(Reporting by Karolos Grohmann; editing by Toby Davis)
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