Cytokine-induced β-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-κB (NF-κB) is activated by interleukin-1β (IL-1β), and its activity promotes the expression of several β-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1β + γ-interferon [IFN-γ])-induced expression of NF-κB in β-cell apoptosis, rat β-cells were infected with the recombinant adenovirus AdIκB^(SA)2, which contained a nondegradable mutant form of inhibitory κB (IκB^(SA)2, with S32A and S36A) that locks NF-κB in a cytosolic protein complex, preventing its nuclear action. Expression of IκB^(SA)2 inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-κB. Cytokine-induced gene expression of several NF-κB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIκB^(SA)2, as established by reverse transcriptase–polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, β-cell survival after IL-1β + IFN-γ treatment was significantly improved by IκB^(SA)2 expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-κB activation, under in vitro conditions, has primarily a pro-apoptotic function in β-cells.