Activated Src have been strongly implicated in the development, progression, and metastasis of many human cancers. Although soy isoflavones exhibit potential anticancer activity, effects of isoflavones on oncogenic activity of Src remains unknown. Using v-src-transfected human adenocarcinoma cells (HAG/src3-1), we investigated the functional role of Src in anti-proliferative activity of isoflavones including genistein, daidzein, glycitein and equol. The growth of HAG/neo3-5 vehicle control cells was inhibited potently by genistein and equol, but modestly by daidzein and glycitein. In contrast, Src activation conferred resistance to either daidzein, glycitein or equol, but rendered the cells more sensitive to genistein, compared to HAG/neo3-5 cells. Genistein significantly arrested HAG/src3-1 cells at G2/M, while neither daidzein, glycitein nor equol arrested the cells at any cell cycle phases. Apoptosis was not induced by either isoflavones. Genistein increased the expression levels of p53 and p21 with decreased phosphorylated p21, but did not affect the levels of major cyclin-CDK complexes. Taken together, genistein would be considered as the only isoflavone component that may potentially suppress Src-driven proliferative activity by arresting at G2/M induction through increasing the p21 levels, thus providing the mechanistic rationale for the potential use of genistein for the prevention of human cancers with activated Src.