This study was designed to examine the effect of genistein, a botanical derived primarily from legumes, on pancreatic β-cell proliferation and the related molecular mechanisms. Diabetes mellitus is a major and growing public health problem worldwide. Both in type 1 (T1D) and type 2 diabetes (T2D), the deterioration of glycemic control over time is primarily caused by an inadequate mass and progressive dysfunction of β-cells. Therefore, the search for novel, safe and cost-effective agents that can enhance islet β-cell proliferation, thereby preserving β-cell mass, could be one of the essential strategies to prevent diabetes, given that β-cells have the potential to regenerate by proliferation of pre-existing b-cells in both physiological condition and after onset of diabetes. Genistein has various biological actions. However, studies on whether genistein has an effect on pancreatic β-cell function are very limited. Our laboratory recently found that genistein activates cAMP/protein kinase A (PKA) signaling in both clonal β-cells and mouse islets. Here I present evidence that genistein induced cellular proliferation of clonal rat pancreatic β-cells (INS1) and human islets following 24 h of incubation. This effect was dose-dependent with 5 µM genistein inducing a maximal 41% increase. The effect of genistein on cell proliferation was not dependent on estrogen receptors because this effect was not blocked by the estrogen receptor inhibitor ICI182,780. In addition, the genistein effect on β-cell proliferation was not shared by 17-β-estradiol or a host of structurally related flavonoid compounds, suggesting that this genistein action is structure-specific. Pharmacological or molecular intervention of PKA or MEK1/2, the upstream kinase of p42/44 mitogen activated protein kinases (ERK1/2), completely abolished the genistein-stimulated proliferation of INS1 cells and human islets, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein increased intracellular cAMP and subsequently activated PKA in human islets. Genistein also caused a rapid and sustained phosphorylation of ERK1/2 with a maximal increase of 185% at 5 µM genistein. The genistein-induced ERK1/2 activation was completely ablated by inhibition of PKA in INS1 cells and human islets. Furthermore, I found that genistein induced protein expression of cyclin D1, a nuclear target of PKA and ERK1/2 activation and a major cell-cycle regulator essential for ï ¢-cell growth. These findings demonstrated that genistein may be a plant-derived growth factor for pancreatic β-cells involving induction of cyclin D1 via activation of the cAMP/PKA-dependent ERK1/2 signaling pathway, thereby providing a novel role for genistein in the regulation of pancreatic β-cell function.