Prolactin is not only a pituitary hormone but an immunoregulatory hormone secreted from lymphocytes. Prolactin induction in relation to hepatitis C virus (HCV) infection has not been elucidated. The serum levels of prolactin were examined in 232 HCV-infected subjects positive for anti-HCV antibody and 65 healthy controls negative for it, who were recruited in the cohort study. The prolactin mRNAs were measured in peripheral blood mononuclear cells (PBMCs) of eleven healthy volunteers including five men and six women before and after stimulation by HCV in vitro. The serum level of prolactin and prolactin mRNA in PBMCs were measured by chemiluminescence immunoassay and real-time PCR, respectively. The serum levels of prolactin were significantly higher in the HCV-infected subjects (median: 7.5, IQR: 5.7–10.9 ng/ml) than in the controls (median: 5.6, IQR: 4.4–8.3 ng/ml) (P < 0.01). They were significantly higher in HCV-infected males (median: 8.0, IQR: 5.9–11.8 ng/ml) than in the controls (median: 4.8, IQR: 4.2–5.9 ng/ml) (P < 0.001), however, the difference was not significant between HCV-infected females (median: 7.3, IQR: 5.6–10.5 ng/ml) and the controls (median: 6.4, IQR: 5.3–9.8 ng/ml). The mRNA expression of prolactin was induced in PBMCs of all males, but it was induced in PBMCs of the two of six females examined in vitro. These results suggest that the serum level of prolactin is higher in HCV-infected males than in healthy males, and that HCV infection induces the mRNA expression of prolactin in PBMCs that is more apparent in male than in females.