In diabetes, the death of insulin-producing β-cells by apoptosis leads to insulin deficiency. The lower prevalence of diabetes in females suggests that female sex steroids protect from β-cell injury. Consistent with this hypothesis, 17β-estradiol (estradiol) manifests antidiabetic actions in humans and rodents. In addition, estradiol has antiapoptotic actions in cells that are mediated by the estrogen receptor-a (ERα), raising the prospect that estradiol antidiabetic function may be due, in part, to a protection of β-cell apoptosis via ERα. To address this question, we have used mice that were rendered estradiol-deficient or estradiol-resistant by targeted disruption of aromatase (ArKO) or ERα (αERKO) respectively. We show here that in both genders, ArKO^−/− mice are vulnerable to β-cell apoptosis and prone to insulin-deficient diabetes after exposure to acute oxidative stress with streptozotocin. In these mice, estradiol treatment rescues streptozotocin-induced β-cell apoptosis, helps sustain insulin production, and prevents diabetes. In vitro, in mouse pancreatic islets and β-cells exposed to oxidative stress, estradiol prevents apoptosis and protects insulin secretion. Estradiol protection is partially lost in β-cells and islets treated with an ERα antagonist and in αERKO islets. Accordingly, αERKO mice are no longer protected by estradiol and display a gender nonspecific susceptibility to oxidative injury, precipitating β-cell apoptosis and insulin-deficient diabetes. Finally, the predisposition to insulin deficiency can be mimicked in WT mice by pharmacological inhibition of ERα by using the antagonist tamoxifen. This study demonstrates that estradiol, acting, at least in part, through ERα, protects β-cells from oxidative injury and prevents diabetes in mice of both genders.