OBJECTIVE: To investigate the molecular mechanism of miR-26b on the proliferation and apoptosis of gastric cancer MGC803 cells. STUDY DESIGN: The expression level of miR-26b was detected by RT-PCR in 50 cases of gastric cancer and normal adjacent tissues, and miR-26b of MGC803 cells was overexpressed by liposome transfection, and the proliferation and apoptosis level of gastric cancer cells were detected. RESULTS: The expression level of miR-26b in gastric cancer was significantly higher than that in adjacent tissues. The overexpression of miR-26b could significantly inhibit the clonogenesis and proliferation of gastric cancer cells and promote the apoptosis of gastric cancer cells. In mechanism, overexpression of miR-26b could bind to the protein of human equilibrative nucleoside transporter 1 (hENT1) and inhibit the expression of RhoA and Rock-1. The antitumor effect of miR-26b on gastric cancer cells in vivo was further confirmed by subcutaneous tumorigenesis experiment. Finally, we found that the protein level of hENT1 in gastric cancer tissue with low expression of miR-26b was significantly higher than that in cancer tissue with high expression of miR-26b. CONCLUSION: The expression level of miR-26b in gastric cancer is significantly decreased. Overexpression of miR-26b can inhibit the proliferation of gastric cancer cells by targeting hENT1 and promoting its apoptosis. (Anal Quant Cytopathol Histpathol 2021;43: 945–953) © Science Printers and Publishers, Inc.