Tumor cells exhibit aberrantly upregulated glycolysis, in which glycolytic enzyme enolase 1 (ENO1) plays a critical role. However, how ENO1 activity is dynamically regulated in tumor cells remains elusive. Here, we demonstrate that hypoxic stimulation promotes the binding of p38 MAP kinase to choline kinase α (CHKα). p38 phosphorylates CHKα, subsequently leading to CHKα to interact with ENO1. Importantly, CHKα functions as a protein kinase and phosphorylates ENO1 thereby enhancing the metabolic activity of ENO1 to increase glycolysis.