AIMS: Previous studies have indicated that vitamin D deficiency correlates with cancer risk and vitamin D potentiates antitumor effects in a variety of cancers. The antitumor effect of vitamin D on gastric cancer was rarely studied. We aimed to investigate the antitumor effect of vitamin D on gastric cancer and underlying mechanisms. MAIN METHODS: We investigated the antitumor activity of the active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on gastric cancer cells (TMK1) and immortalized normal gastric cells (HFE145) by using MTT, colony formation and Flow cytometry assays. We demonstrated the important role of vitamin D receptor (VDR) and mutant p53 (mutp53) in mediating the antitumor action of 1,25(OH)2D3 by using siRNA, western-blot, immunofluorescent staining and immunoprecipitation assays. KEY FINDINGS: 1,25(OH)2D3 could significantly inhibit proliferation and induce cell cycle arrest in TMK1 but not in HFE145. Furthermore, 1,25(OH)2D3 stimulated p21 expression and suppressed cyclin-dependent kinase 2 (CDK2) expression in TMK1 in a VDR-dependent manner. High levels of VDR in human gastric cancer tissues and cancer cell lines implicated that vitamin D could display more potent pharmacological action against malignant cells. Besides, mutp53 but not wild type p53 was essential for 1,25(OH)2D3-stimulated upregulation of p21 in gastric cancer cells. Indeed, mutp53 could stabilize nuclear VDR level through interaction with VDR. SIGNIFICANCE: Our results suggest that 1,25(OH)2D3 inhibits gastric cancer cell growth through VDR and mutp53 interaction followed by the modulation of p21/CDK2. We propose that vitamin D might be used for the prophylactic treatment for malignant diseases in the stomach.