Natural killer (NK) cells act as innate immune cells against hepatitis C virus (HCV) infection. Interferon-α (IFN-α) and ribavirin are the standard treatments for patients with HCV infection. This study is aimed at investigating the dynamic changes in the frequency of different subsets of NK cells following treatment in xx chronic hepatitis C (CHC) patients. CHC patients were treated with peg-IFN or IFN-α, and followed up for 72 weeks. The frequency of different subsets of NK in CHC patients was determined longitudinally by flow cytometry. Treatment with the standard therapy increased the percentages of NKp30(+), NKp46(+), and CD107a(+) NK cells, which were positively correlated with the declining of serum HCV-RNA, but not IFN-γ(+) NK cells. NKG2A(+) and KIR2DL3(+) NK cells were associated with an early virological response in CHC patients. Treatment with IFN-α adjusts the balance of activated receptors and inhibitory receptors and enhances the cytotoxic activity of NK cells. Therefore, measuring NK subsets may be valuable for therapeutic responses in CHC patients.