Patients with hepatitis C virus (HCV) infection have a greater risk of developing type 2 diabetes mellitus. However, the mechanism of this association is unclear. In this study, we examined the potential defects in upstream insulin signaling pathways in liver specimens obtained from nonobese/nondiabetic subjects with HCV infection. Fasting liver biopsy specimens were obtained from 42 HCV-infected subjects and 10 non-HCV-infected subjects matched for age and body mass index. Liver tissues were exposed to insulin and examined for the contents and phosphorylation/activation status of the upstream insulin signaling molecules by immunoprecipitation and Western blot analysis. HCV infection resulted in a trend toward a 2-fold to 3-fold increase in insulin receptor (IR) and insulin receptor substrate (IRS)-1 contents when compared with non-HCV. In contrast, insulin-stimulated IRS-1 tyrosine phosphorylation was decreased by 2-fold in HCV-infected subjects compared with non-HCV-infected subjects (P < .05). The observed reductions in IRS-1 tyrosine phosphorylation were accompanied by a 3.4-fold decrease in IRS-1/p85 phosphatidylinositol 3-kinase (PI3-kinase) association and a 2.5-fold decrease in IRS-1-associated PI3-kinase enzymatic activity (P < .05 vs. non-HCV). This was accompanied by a marked reduction in insulin-stimulated Akt phosphorylation without any alterations in mitogen-activated protein kinase (MAPK) phosphorylation. Cellular contents of the hepatic p85 subunit of PI3-kinase were comparable between HCV-infected and non-HCV-infected subjects. In conclusion, we found that (1) HCV infection leads to a postreceptor defect in IRS-1 association with the IR and (2) insulin signaling defects in hepatic IRS-1 tyrosine phosphorylation and PI3-kinase association/activation may contribute to insulin resistance, which leads to the development of type 2 diabetes mellitus in patients with HCV infection.