We investigated the effect of Artesunate on cell proliferation, cell cycle, apoptosis and the PTEN/FAK signaling pathway on the RPMI8226 cell line in vitro. After treatment with different concentration of ART, the proliferation of RPMI8226 cells was significantly inhibited. The cell apoptotic rate was increased in a dose-dependent manner. Cell cycle analysis showed that the proportion of cells in G(0)/G(1) phase decreased, while the proportion of cells in G(2)/M phases increased significantly as compared to that in untreated cells. The mRNA and protein expression levels of cyclin B1, PTEN, caspase-3/7 significantly increased, while survivin, CDC2 and FAK significantly decreased when compared with the untreated control group. These results indicate that ART can inhibit RPMI8226 cells proliferation and trigger cell apoptosis with G(2)/M arrests, which may occur through up-regulating caspase-3, caspase-7, cyclin B1, and PTEN expression while inhibiting survivin, CDC2, and FAK expression.