Suppressor of cytokine signaling 3 (SOCS-3) is a negative feedback regulator of IFN-γ signaling, shown up-regulated in mouse bone marrow cells by the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IFN-γ. IL-1β and IFN-γ alone, or potentiated by TNF-α, are cytotoxic to the insulin producing pancreatic β-cells and β-cell lines in vitro and suggested to contribute to the specific β-cell destruction in Type-1 diabetes mellitus (T1DM). Using a doxycycline-inducible SOCS-3 expression system in the rat β-cell line INS-1, we demonstrate that the toxic effect of both IL-1β or IFN-γ at concentrations that reduced the viability by 50% over 3 days, was fully preventable when SOCS-3 expression was turned on in the cells. At cytokine concentrations or combinations more toxic to the cells, SOCS-3 overexpression yielded a partial protection. Whereas SOCS-3-mediated inhibition of IFN-γ signaling is described in other cell systems, SOCS-3 mediated inhibition of IL-1β signaling has not previously been described. In addition we show that SOCS-3 prevention of IL-1β-induced toxicity is accompanied by inhibited transcription of the inducible nitric oxide synthase (iNOS) by 80%, resulting in 60% decreased formation of the toxic nitric oxide (NO). Analysis of isolated native rat islets exposed to IL-1β revealed a naturally occurring but delayed up-regulated SOCS-3 transcription. Influencing SOCS-3 expression thus represents an approach for affecting cytokine-induced signal transduction at a proximal step in the signal cascade, potentially useful in future therapies aimed at reducing the destructive potential of β-cell cytotoxic cytokines in T1DM, as well as other cytokine-dependent diseases.