It has been reported that Harmine hydrochloride (HH) has an inhibitory effect on tumor cells, but the effect and mechanism of HH on gastric cancer cells remains unclear. The aim of this study was to investigate the effect and mechanism of HH on human gastric cancer cell line. In present study, results showed that HH could inhibit AGS, SGC7901 and HGC-27 cells in a time-dose-dependent manner (P < 0.01). Furthermore, this study demonstrated that more cells were arrested in G0/G1 phase, and apoptosis rate of AGS cells was significantly increased after HH treatment (P < 0.01). In addition, the study results showed that the mRNA and proteins of CyclinE, CyclinD1, PCNA declined dramatically, while p27, p21 increased significantly (P < 0.01). The results in this research also showed that the mRNA and proteins of Survivin and Bcl-2 decreased, while the expression of Bax, caspase-3, Bad increased significantly (P < 0.01). Also, the results of this study showed that invasion and migration of AGS cells decreased significantly after HH treatment (P < 0.01), with the expression of MMP-2, HIF-1 alpha and PRDX1 decreasing on observation after HH treatment (P < 0.01). In conclusion, HH has the property to inhibit GC cells via regulating GC cells' proliferation, apoptosis, invasion and migration.