Transplantation of islets of Langerhans is a potential cure for type 1 diabetes, but its success is hampered by destruction of the islets. The data presented herein suggest that the active metabolite of vitamin D3 [1,25-(OH)2D3] may promote islet cell survival by modulating the effects of inflammatory cytokines, which contribute to beta-cell demise. We investigated some of the mechanisms triggering the apoptotic machinery in rat insulinoma RINm5F cells and human islets treated with IL-1beta plus interferon-gamma plus TNFalpha and assessed the effects of 1,25-(OH)2D3 in these processes. Mitochondrial transmembrane permeability and apoptotic features, determined by percentage of sub-G1 cells, quantitation of DNA strand breaks, and Hoechst staining, were significantly increased by cytokines and reverted toward control values by 1,25-(OH)2D3 cotreatment. The cytoprotection of cells correlated with the abrogation of cytokine-induced nitric oxide production. The activation of nuclear factor-kappaB plays a key role in the different pathways implicated in nitric oxide generation. We demonstrated for the first time, in both RINm5F cells and human islets, that 1,25-(OH)2D3 was able to induce and maintain high levels of A20, an antiapoptotic protein known to block nuclear factor-kappaB activation. Our study showed a clear efficiency of 1,25-(OH)2D3 on the apoptotic machinery triggered by cytokines in beta-cells and suggests that 1,25-(OH)2D3 could help overcome a major obstacle encountered in the cellular therapy of diabetes, such as nonfunction in the immediate posttransplantation period.