Despite extensive studies on the antitumor properties of berberine, a small molecule derived from Coptidis rhizoma (Huanglian in Chinese) and many other plants, the underlying mechanism remains poorly understood. Here, we found that berberine-induced cell apoptosis in human gastric cancer cells with the increase of the expression level of cleaved poly ADP-ribose polymerase and caspase-3, and the impairment of mitochondrial membrane potential (Δψm) in berberine-treated gastric cancer cells. In our further studies, the results demonstrated that Akt-related mitochondrial pathway may partly involve in the berberine-induced apoptosis in human gastric cancer cells. Moreover, berberine inhibited the Akt/mTOR/p70S6/S6 pathway in berberine-treated BGC-823 cells. Meanwhile, berberine significantly inhibited the activation of Akt and suppressed tumor growth in xenograft nude mice injected with human gastric cancer cells. Thus, our findings reveal that the underlying mechanism that Akt signaling may contribute to berberine-induced cell apoptosis in gastric cancer cells and might represent an important molecular basis for berberine to act as an anticancer agent.