Genistein, a major isoflavone found in soybeans, exhibits anticarcinogenic properties. The inhibitory effect of genistein on cell proliferation is associated with G2/M cell cycle arrest and inhibition of cdc2 activities. Here we assessed the role of PTEN in regulation of genistein-mediated G2/M cell cycle arrest in the gastric cancer cell lines (SGC-7901 and BGC-823). After 24 h following treatment, genistein induced a concentration-dependent accumulation of cells in the G2/M phase of the cell cycle. The sustained G2/M arrest by genistein in SGC-7901 and BGC-823 cells is associated with increased phospho-cdc2 (Tyr15) and decreased cdc2 protein. Genistein treatment increased Wee1 levels and decreased phospho-Wee1 (Ser 642). Moreover, genistein substantially decreased the Ser473 and Thr308 phosphorylation of Akt and upregulated PTEN expression. Downregulation of PTEN by siRNA in genistein-treated cells increased phospho-Wee1 (Ser642), whereas decreased phospho-Cdc2 (Tyr15), resulting in decreased the G2/M cell cycle arrest. Therefore, induction of G2/M cell cycle arrest by genistein involved upregulation of PTEN.