CD8(+)CD28(-) regulatory T cells (Tregs) play important roles in chronic viral infections. Programmed death 1 (PD-1) is highly expressed on hepatitis C virus (HCV)-specific CTLs. However, little is known regarding the role of CD8(+)CD28-PD1(+) T cells in hepatitis C. Herein, we found that the frequency of CD8(+)CD28(-)PD1(+), but not CD8(+)CD28(-)PD1(-) T cells, correlated with markers of chronic hepatitis C virus (HCV) infection and the response to treatment. Our results showed that CD8(+)CD28-PD1(+) T cells were significantly elevated in chronic HCV-infected patients and there was a distinct correlation between the frequency of CD8(+)CD28-PD1(+) T cells and serum levels of HCV RNA. During a 48-week course of treatment with peg-IFN-a2a plus ribavirin, dynamic changes in the frequencies of CD8(+)CD28-PD1(+) T cells were observed, associated with the virologic response. IL-10 secretion may explain the suppressive function of CD8(+)CD28-PD1(+) T cells in chronic HCV-infected patients. Overall, our study demonstrates that PD-1 is an important marker of CD8(+)CD28(-) Tregs in chronic HCV infection. Thus, the frequency and regulatory function of CD8(+)CD28-PD1(+) T cells play vital roles in HCV infection and the response to treatment.