Hepatitis C virus (HCV) perturbs the host's lipid metabolism and often results in hepatic steatosis. In nonalcoholic fatty liver disease, the intrahepatic down-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a critical mechanism leading to steatosis and its progression toward fibrosis and hepatocellular carcinoma. However, whether an HCV infection triggers the formation of large lipid droplets through PTEN-dependent mechanisms is unknown. We assessed PTEN expression in the livers of patients infected with HCV genotype 1 or 3 with or without steatosis. The role of PTEN in the HCV-induced biogenesis of lipid droplets was further investigated in vitro with hepatoma cells transduced with the HCV core protein of genotype 1b or 3a. Our data indicate that PTEN expression was down-regulated at the posttranscriptional level in steatotic patients infected with genotype 3a. Similarly, the in vitro expression of the HCV genotype 3a core protein (but not 1b), typically leading to the appearance of large lipid droplets, down-regulated PTEN expression by a mechanism involving a microRNA-dependent blockade of PTEN messenger RNA translation. PTEN down-regulation promoted in turn a reduction of insulin receptor substrate 1 (IRS1) expression. Interestingly, either PTEN or IRS1 overexpression prevented the development of large lipid droplets, and this indicates that the down-regulation of both PTEN and IRS1 is required to affect the biogenesis of lipid droplets. However, IRS1 knockdown per se did not alter the morphology of lipid droplets, and this suggests that other PTEN-dependent mechanisms are involved in this process. CONCLUSION: The down-regulation of PTEN and IRS1 is a critical event leading to the HCV genotype 3a-induced formation of large lipid droplets in hepatocytes.