Human hepatocellular carcinoma (HCC) remains incurable with current therapies, and novel biologically based therapies are urgently needed. Arsenic agents have long been used as anticancer agents in traditional Chinese medicine. In this study, to evaluate the effect of As2O3 on HCC cells, we investigate cell growth inhibition, cell cycle arrest, and the molecular mechanism after As2O3 treatment in human HCC cells in vitro. We detected the proliferation of HCC cells by the Cell Counting Kit and FACS/Calibur Flow Cytometer and analyzed the expression and localization of FOXO3a by Western blotting Analysis and Cell Fractionation. Furthermore, we study the Akt activation after As2O3 treatment and the HCC cells proliferation after combination of As2O3 with PI3K inhibitor Wortmannin. As2O3 significantly inhibited the proliferation of all the three HCC cell lines (SMMC7721, HepG2, Hep3B) tested in this study in a dose-dependent manner. Western blotting revealed that treatment HCC cells HepG2 with As2O3 resulted in the increasing of FOXO3a expression and triggered phosphorylation of FOXO3a at the Thr32 residue decrease. This FOXO3a accumulation correlated well with the As2O3-induced reduction of active Akt. Nuclear and cytoplasmic protein extracts isolated from the HCC cell line HepG2 revealed that the amount of nuclear FOXO3a was increased by treatment with As2O3, whereas the amount of cytoplasmic FOXO3a was decreased. Both As2O3 and PI3K/Akt inhibitor Wortmannin induced cell cycle arrest. However, compared with As2O3 alone, PI3K inhibitor Wortmannin combined with As2O3 enhanced the antitumor effect of As2O3 through induction of apoptosis. These findings suggest that As2O3 at a clinically safe concentration may be an effective chemotherapeutic agent, and that As2O3 and PI3K/Akt inhibitor Wortmannin may synergize for HCC cells. Taken together, the present study may suggest a specific molecular mechanism by which HCC cell lines are susceptible to the As2O3 therapy through FOXO3a expression and localization.