Introduction. Proinflammatory cytokines (PIC) (interleukin-1β, interferon-γ, and tumor necrosis factor α) are released after intraportal islet transplantation lead to functional suppression and islet apoptosis. Estradiol has been shown to promote survival of cells undergoing PIC-induced apoptosis. In this study, we evaluated the effects of estradiol on isolated human pancreatic islet (IHPI) survival after exposure to PIC and analyzed potential mechanisms of action. Methods. Hand-picked, freshly isolated IHPI were incubated with PIC and estradiol. Viability was analyzed from single islet cells stained with ethidium bromide and acridine orange, apoptosis using a quantitative kit, NF-κB nuclear translocation using a promoter-Luciferase NF-κB responsive construct, mitochondrial permeability transition using the ApoAlert Mitochondrial kit, and caspase 9 by a fluorometric assay. In vitro functionality was examined by static incubation, and a limited number of islets were transplanted in nonobese diabetic, severe combined immunodeficient mice. Results. 17β-Estradiol induced a dose-dependent increase in islet viability, an effect partially reversed by the estrogen receptor antagonist ICI 182,780. In vitro, islets treated with estradiol presented higher stimulation index. Euglycemia was achieved in 6 of 12 animals that received estradiol-treated islets compared with 1 of 12 control animals. Lower NF-κB nuclear translocation, cytochrome c release, and caspase 9 activation occurred in islets treated with estradiol. Conclusions. Estradiol promoted IHPI survival and improved functionality after PIC exposure in vitro and in vivo after transplantation. The molecular mechanisms involved included a decrease in NF-κB nuclear translocation, decrease in mitochondrial cytochrome c release, and caspase 9 activation. The use of estradiol might be beneficial in clinical islet transplantation.