The protective effect of KIOM-4, a mixture of plant extracts, was examined against streptozotocin (STZ)-induced mitochondrial oxidative stress in rat pancreatic β-cells (RINm5F). KIOM-4 scavenged superoxide and hydroxyl radicals generated by xanthine/xanthine oxidase and Fenton reaction (FeSO(4)/H(2)O(2)), respectively, in a cell-free chemical system. In addition, a marked increase in mitochondrial reactive oxygen species (ROS) was observed in STZ-induced diabetic cells; this increase was attenuated by KIOM-4 treatment. Mitochondrial manganese superoxide dismutase (Mn SOD) activity and protein expression were down-regulated by STZ treatment and up-regulated by KIOM-4 treatment. In addition, NF-E2 related factor 2 (Nrf2), a transcription factor for Mn SOD, was up-regulated by KIOM-4. KIOM-4 prevented STZ-induced mitochondrial lipid peroxidation, protein carbonyl and DNA modification. Moreover, KIOM-4 treatment restored the loss of mitochondrial membrane potential (Δψ) that was induced by STZ treatment, and inhibited the translocation of cytochrome c from the mitochondria to the cytosol. In addition, KIOM-4 treatment elevated the level of ATP, succinate dehydrogenase activity and insulin level, which were reduced by STZ treatment. These results suggest that KIOM-4 exhibits a protective effect through its antioxidant effect and the attenuation of mitochondrial dysfunction in STZ-induced diabetic cells.