Anthocyanins are naturally occurring plant pigments and exhibit an array of pharmacological properties. Our previous study showed that black rice pigment extract rich in anthocyanin prevents and ameliorates high-fructose-induced insulin resistance in rats. In present study, cyanidin 3-glucoside (Cy-3-G), a typical anthocyanin most abundant in black rice was used to examine its protective effect on insulin sensitivity in 3T3-L1 adipocytes exposed to H2O2 (generated by adding glucose oxidase to the medium) or tumor necrosis factor α (TNF-α). Twelve-hour exposure of 3T3-L1 adipocytes to H2O2 or TNF-α resulted in the increase of c-Jun NH2-terminal kinase (JNK) activation and insulin receptor substrate 1 (IRS1) serine 307 phosphorylation, concomitantly with the decrease in insulin-stimulated IRS1 tyrosine phosphorylation and cellular glucose uptake. Blocking JNK expression using RNA interference efficiently prevented the H2O2- or TNF-α-induced defects in insulin action. Pretreatment of cells with Cy-3-G reduced the intracellular production of reactive oxygen species, the activation of JNK, and attenuated H2O2- or TNF-α-induced insulin resistance in a dose-dependent manner. In parallel, N-acetyl-cysteine, an antioxidant compound, did not exhibit an attenuation of TNF-α-induced insulin resistance. Taken together, these results indicated that Cy-3-G exerts a protective role against H2O2- or TNF-α-induced insulin resistance in 3T3-L1 adipocytes by inhibiting the JNK signal pathway.