Loss of pancreatic β-cells by oxidative stress or cytokines is associated with diabetes mellitus (DM). DJ-1 is known to as a multifunctional protein, which plays an important role in cell survival. To investigate the effects of DJ-1 against combined cytokine (IL-1β, IFN-γ and TNF-α)-induced RINm5F cell death, we prepared cell permeable wild type (WT) and mutant type (M26I) Tat-DJ-1 proteins. Both Tat-DJ-1 proteins transduced into RINm5F cells. WT Tat-DJ-1 proteins significantly protected against cell death from cytokines by reducing intracellular toxicities. Also, WT Tat-DJ-1 proteins markedly regulated cytokines-induced pro- and anti-apoptosis proteins. However, M26I Tat-DJ-1 protein showed relatively low protective effects compared to WT Tat-DJ-1 protein. Our experiments demonstrated that WT Tat-DJ-1 protein protects against cytokine-induced RINm5F cell death by suppressing intracellular toxicities and regulating apoptosis-related protein expression, suggesting that WT Tat-DJ-1 protein could potentially serve as a therapeutic agent for DM and cytokine related diseases.