Type 1 diabetes mellitus (T1DM) is caused by the selective destruction of insulin-producing beta-cells. This process is mediated by cells of the immune system through release of nitric oxide, free radicals and pro-inflammatory cytokines, which induce a complex network of intracellular signalling cascades, eventually affecting the expression of genes involved in beta-cell survival. The aim of our study was to investigate possible mechanisms of resistance to cytokine-induced beta-cell death. To this purpose, we created a cytokine-resistant beta-cell line (beta-TC3R) by chronically treating the beta-TC3 murine insulinoma cell line with IL-1beta + IFN-gamma. beta-TC3R cells exhibited higher proliferation rate and resistance to cytokine-mediated cell death in comparison to the parental line. Interestingly, they maintained expression of beta-cell specific markers, such as PDX1, NKX6.1, GLUT2 and insulin. The analysis of the secretory function showed that beta-TC3R cells have impaired glucose-induced c-peptide release, which however was only moderately reduced after incubation with KCl and tolbutamide. Gene expression analysis showed that beta-TC3R cells were characterized by downregulation of IL-1beta and IFN-gamma receptors and upregulation of SOCS3, the classical negative regulator of cytokines signaling. Comparative proteomic analysis showed specific upregulation of 35 proteins, mainly involved in cell death, stress response and folding. Among them, SUMO4, a negative feedback regulator in NF-kB and JAK/STAT signaling pathways, resulted hyper-expressed. Silencing of SUMO4 was able to restore sensitivity to cytokine-induced cell death in beta-TC3R cells, suggesting it may play a key role in acquired cytokine resistance by blocking JAK/STAT and NF-kB lethal signaling. In conclusion, our study represents the first extensive proteomic characterization of a murine cytokine-resistant beta-cell line, which might represent a useful tool for studying the mechanisms involved in resistance to cytokine-mediated beta-cell death. This knowledge may be of potential benefit for patients with T1DM. In particular, SUMO4 could be used as a therapeutical target.