Autophagy is closely associated with tumorigenesis and the response of tumor cells to chemotherapeutic drugs. Matrine has a wide range of pharmacological effects including antitumor activity in vitro and in vivo. The aim of this study was to determine the significance of autophagy in antineoplastic effects of matrine and to further elucidate the molecular mechanism by which matrine induces autophagy in gastric cancer cells. The results showed that matrine significantly inhibited the proliferation of gastric cancer SGC-7901 cells and induced G1-phase cell cycle arrest. Furthermore, both autophagy and apoptosis were activated during the matrine-induced death of SGC-7901 cells. Propidium iodide staining demonstrated that autophagic inhibitor 3-methyladenine (3-MA) or bafilomycin A1 enhanced lethality of matrine against gastric cancer cells. Moreover, after pretreatment with 3-MA, some of gastric cancer cells treated with matrine exhibited prototypical characteristics of apoptosis by transmission electron microscopy. The ability of 3-MA to increase matrine-induced apoptosis was further confirmed by Annexin V-FITC/PI staining. Also, the combination of matrine and 3-MA was more potent than matrine alone in inhibiting the proliferation of SGC-7901 cells assessed by sulphorhodamine B assay. Furthermore, administration of the pan-caspase inhibitor zVAD-fmk or autophagy inducer rapamycin decreased the matrine-induced cell death. In addition, western blot analysis showed that matrine treatment did not inhibit the phosphorylation of Akt and its downstream effectors mammalian target of rapamycin (mTOR) as well as p70 ribosomal protein S6 kinase (p70S6K), although the levels of the total Akt and mTOR were decreased. RT-PCR revealed that Beclin 1 is involved in matrine-induced autophagy and the pro-apoptotic mechanisms of matrine may be associated to its upregulation on Bax expression. These findings indicate that matrine is a potent antitumor agent for treating gastric cancer. Autophagy was activated as a protective mechanism against matrine-induced apoptosis and inhibition of autophagy may be an attractive strategy for enhancing the antitumor potential of matrine in gastric cancer.