Depression is known to occur frequently in chronic hepatitis C viral (HCV) patients receiving interferon (IFN)-α therapy. In this study, we investigated whether indoleamine 2,3-dioxygenase1 (IDO1)-mediated tryptophan (TRP) metabolism plays a critical role in depression occurring as a side effect of IFN-α therapy. Increases in serum kynurenine (KYN) and 3-hydroxykynurenine (3-HK) concentrations and in the ratios of KYN/TRP and 3-HK/kynurenic acid (KA) were much larger in depressive HCV patients than in non-depressed patients following therapy. Furthermore, transfection of a plasmid continuously expressing murine IFN-γ into normal mice significantly increased depression-like behavior. IFN-γ gene transfer also resulted in a decrease in serum TRP levels in the mice while KYN and 3-HK levels were significantly increased in both serum and frontal cortex. Genetic deletion of IDO1 in mice abrogated both the increase in depression-like behavior and the elevation in TRP metabolites' levels, and the turnover of serotonin in the frontal cortex after IFN-γ gene transfer. These results indicate that the KYN pathway of IDO1-mediated TRP metabolism plays a critical role in depressive symptoms associated with IFN-α therapy.