AbstractChemoresistance remains a major challenge for the treatment of glioma. In this study, we investigated the role of Clock 1 (Clk1), which encodes an enzyme that is necessary for ubiquinone biosynthesis in glioma chemoresistance in vitro. The results showed that Clk1 was highly expressed in GL261 mouse glioma cells which were most sensitive to 1,3Bis (2‐chloroethyl) 1 nitrosourea (BCNU) while was low expressed in BCNU resistant cells such as glioma cancer stem cells, T98G, U87MG and U251 glioma cells. Knockdown of Clk1 in GL261 glioma cells significantly reduced BCNU‐ or cisplatin‐induced cell apoptosis, whereas the proliferative activity and the expression of multidrug resistance‐related genes including MDR1, O6‐methylguanine‐DNA methyltransferase, and GSTP1 were not changed. When Clk1 was re‐expressed in Clk1 knockdown GL261 glioma cells, the BCNU sensitivity was restored. The mechanistic study revealed that knockdown of Clk1 in GL261 glioma cells increased aerobic glycolysis including high glucose consumption, lactate production, and up‐regulation of glycolysis‐associated genes. Inhibition of glycolysis can reverse the chemoresistance elicited by Clk1 knockdown in GL261 cells. Moreover, knockdown of Clk1 induced HIF‐1α expression in GL261 glioma cells which was found to be mediated by AMP‐activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) signaling pathway. Both metformin and rapamycin reversed the chemoresistance of Clk1 knockdown GL261 glioma cells. Over‐expression of Clk1 significantly increased the sensitivity of T98G or U251 human glioblastoma cells to BCNU which was accompanied by decreased lactate secretion, decreased expression of HIF‐1α, AMPK activation, and inhibition of mTOR pathway. Inhibition of glycolysis or activation of AMPK did not alter Clk1 expression in variant glioma cell lines suggesting that aerobic glycolysis is not an upstream event of Clk1 expression in glioma cells. Taken together, our results revealed, for the first time, that mitochondrial Clk1 regulated chemoresistance in glioma cells through AMPK/mTOR/HIF‐1α mediated glycolysis pathway. image