-equol, which is enantioselectively produced from daidzein by gut microbiota, has been suggested as a chemopreventive agent against type 2 diabetes mellitus (T2DM), but the underlying mechanisms remain unclear. We investigated the effects of -equol on pancreatic β-cell function. β-Cell growth and insulin secretion were evaluated with male Institute of Cancer Research mice and isolated pancreatic islets from the mice, respectively. The mechanisms by which -equol stimulated β-cell response were examined in INS-1 β-cells. The effect of-equol treatment on β-cell function was assessed in low-dose streptozotocin-treated mice. -equol was used at 10 μmol/L for in vitro and ex vivo studies and was administered by oral gavage (20 mg/kg, 2 times/d throughout the experimental period) for in vivo studies.-equol administration for 7 d increased Ki67-positive β-cells by 27% ( < 0.01) in mice. -equol enantioselectively enhanced glucose-stimulated insulin secretion in mouse pancreatic islets by 41% ( < 0.001). In INS-1 cells, -equol exerted stronger effects than daidzein on cell growth, insulin secretion, and cAMP-response element (CRE)-mediated transcription. These-equol effects were diminished by inhibiting protein kinase A. The effective concentration of -equol for stimulating cAMP production at the plasma membrane was lower than that for phosphodiesterase inhibition.-equol-stimulated CRE activation was negatively controlled by the knockdown of G-protein α subunit group S (stimulatory) and positively controlled by that of G-protein-coupled receptor kinase-3 and -6. Compared with vehicle-treated controls, -equol gavage treatment resulted in an increase in β-cell mass of 104% ( < 0.05), a trend toward high plasma insulin concentrations (by 118%; = 0.06), and resistance to hyperglycemia after streptozotocin treatment (78% of AUC after glucose challenge; < 0.01). -equol administration significantly increased the number of Ki67-positive proliferating β-cells by 62% ( < 0.01) and decreased that of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic β-cells by 75% ( < 0.05). Our results show that -equol boosts β-cell function and prevents hypoglycemia in mice, suggesting its potential for T2DM prevention.