Objective: To investigate the effects of histone deacetylase 3 inhibitor RGFP966 on proliferation and cell cycle of gastric cancer cells and its mechanism. Methods: The activity of MN-45 and MGC-803 cell lines treated with RGFP966 was detected by CCK-8 method.The effect of RGFP966 on the cytoclonal ability of gastric cancer cells was detected by cell cloning assay.Cell cycle was detected by flow cytometry.The expressions of Ki-67, c-Myc, CyclinA2, CyclinD1 and PI3K/AKT signaling pathway were detected by Westernblot assay. Results: Compared with the control group, RGFP966 could significantly inhibit the proliferation and cloning ability of MSN-45 and MGC-803 gastric cancer cells.Compared with the control group, flow cytometry showed that RGFP966 blocked the cell cycle in the G0/G1 phase.Compared with the control group, proliferation-related proteins Ki-67 and c-Myc were decreased after treatment with RGFP966, and the expressions of CyclinA2 and CyclinD1 were decreased. Further results showed that RGFP966 could significantly reduce the phosphorylation levels of PI3K and AKT. Conclusion: RGFP966 inhibits the proliferation of gastric cancer cells and blocks the cycle in G0/G1 phase, which may be related to the inhibition of PI3K/AKT signaling pathway by RGFP966.