Abstract: Objective To investigate the apoptosis effect of miR-26a/b by regulating p53/MDM2 pathway in gastric cancer cells. Methods The expression of miR-26a/b in gastric cell lines （MGC803， MKN-45 and MKN-28） was detected by real-time PCR. The luciferase activity was analyzed to determine the binding of miR-26a/b to MDM2 3’ untranslated region （3’ UTR）. The miR-26a/b precursor molecule mimics and a scramble sequence were transfected into MKN-45 cells， and miR-26a/b inhibitors were transfected into GES-1 cells， respectively. The expression of MDM2， p53 and its downstream genes p21 and Bcl2 were detected by Western blotting. The proliferation rates of these cells were detected by MTT assay after 24， 48， 72 and 96 h. The apoptosis of cells were detected by AnnexinⅤ/PI. Results Compared with GES-1 cells， miR-26a/b was downregulated in gastric cancer cell lines， especially in MKN-45 cells. Luciferase activity analysis showed that the overexpression of miR-26a/b suppressed MDM2 3’UTR reporter vector （Wild） luciferase activity， whereas luciferase activity had no significant change in the 3’UTR mutant reporter vectors. The miR-26a/b inhibited the expression of MDM2 and enhanced the expression of p53 and downstream genes in MKN-45 cells. Inhibition of miR-26a/b enhanced the expression of MDM2， and decreased the expression of p53 and downstream genes in GES-1 cells. The Results of MTT assay showed that miR-26a/b overexpression significantly inhibited the proliferation of MKN-45 cells， while miR-26a/b inhibitor significantly promoted the cell proliferation of GES-1 cells. The AnnexinⅤ/PI assay found that the apoptosis rates of MKN-45 which overexpressed miR-26a/b were significantly higher than control （P＜0.01）. Conclusion miR-26a/b can specifically bind the 3’UTR of MDM2 and disrupt proliferation and apoptosis of gastric cancer cells by regulating p53/MDM2 pathway.