Interleukin-35 (IL-35), secreted mainly by T-regulatory cells (T-regs), has been considered to have immunosuppressive actions in many auto-immune diseases and tumors. However, information about its role in chronic hepatitis C (CHC) infection is still limited. We aimed to study the role of IL-35 within CHC infection and to assess its correlation with T-regs and T-helper 17 cells (Th-17). Therefore, we measured serum IL-35 concentrations using ELISA assay in 25 normal controls (NCs) and in 30 CHC patients before receiving direct antiviral agents (DAA) treatment and after 3 months of treatment end. T-regs and Th-17 cells frequencies were assessed via flow-cytometry in control group and patients’ group before treatment. The results showed that serum IL-35 levels revealed a highly significant increase in CHC patients compared to NCs (P <0.001). Moreover, IL-35 levels significantly decreased in patients 3 months after treatment end (P =0.02). Both Th-17 and T-regs were significantly increased in patients more than in NCs and a positive correlation was observed between them. However, T-regs/Th-17 ratio did not show significant difference from the ratio in NCs. IL-35 levels were positively correlated with viral load and T-regs frequency, but not with Th-17 frequency. IL-35 levels did not correlate with liver enzymes or functions. These results suggested that IL-35 enhances the immunosuppressive functions of T-regs, protecting the liver from HCV induced damage and contributes to viral persistence. IL-35 may represent a possible immunotherapeutic strategy for chronic persistent infection if given with DAA, especially in relapsing or non-responding cases.