The imbalance of regulatory T cells (Tregs) and T helper 17 (Th17) cells contributes to the persistent hepatitis C virus (HCV) infection. However, modulatory factors associated with Tregs-Th17 balance were not fully elucidated. Arecent study demonstrated an immunoregulatory strategy by inactivation of Notch signaling to reverse the disequilibrium of Tregs-Th17 cells in immune thrombocytopenia. Thus, the aim of this study was to assess the effect of Notch signaling in regulating the functions of Tregs and Th17 cells in chronic hepatitis C. A total of 46 patients with chronic hepatitis C and 17 normal controls (NCs) were enrolled. mRNA expressions of Notch1 and Notch2 were semiquantified by real-time reserve polymerase chain reaction. Percentages of Tregs-Th17, levels of key transcriptional factors, and cytokine productions were measured in response to treatment by DAPT, a gamma-secretase inhibitor to suppress Notch signaling. We found that Notch1 and Notch2 mRNAs were significantly elevated in peripheral blood mononuclear cells from chronic hepatitis C patients compared with those from NCs. DAPT treatment reduced Th17 response by downregulation of ROR gamma t expression and interleukin (IL)-17/IL-22 secretion. Tregs proportion, FoxP3 expression, and IL-10 production did not change significantly with DAPT treatment in chronic hepatitis C; however, blockage of Notch signaling inhibited the suppressive function of Tregs. Moreover, effective anti-HCV therapy not only reduced Notch1 and Notch2 expression but also decreased Tregs and Th17 proportions. The current data provided a novel mechanism underlying the modulation of Treg-Th17 balance. The link between Notch signaling and Th cells might lead to a new intervention for breaking immunotolerance of chronic HCV infection.