Gastric cancer is one of the most common malignancy with a leading mortality rate worldwide. Despite the progress in the diagnosis and therapeutic strategy, the associated mortality is still growing. It is of great significance to understand molecular mechanisms of the development of gastric cancer. Glycolysis is a main source of ATP provision for cancer cells including gastric cancer, and targeting glycolysis is a promising therapeutic strategy. Centromere protein U (CENPU) has been found to be overexpressed in many types of cancer. Downregulation of CENPU suppresses the proliferation and invasion of cancer cells. High mobility group box 2 (HMGB2) is identified as a biomarker to diagnose of gastric cancer. Knockdown of HMGB2 inhibits proliferation and glycolysis in gastric cancer cells. In this work, we identified that CENPU was upregulated in gastric cancer. Knockdown of CENPU was able to suppress the proliferation and glycolysis of gastric cancer cells. Further the results showed that the anti-cancer effect of CENPU was HMGB2-dependent. Taken together, CENPU is an upstream factor of HMGB2, which regulates proliferation and glycolysis of gastric cancer.