The immunomodulatory role of natural killer (NK) cells has been recognized recently, but its effects on CD4(+)CD25(+) regulatory T cells (Tregs) during chronic hepatitis B (CHB) infection and treatment remain unclear. A total of 116 nucleos(t)ide analogue (NA)-treated CHB patients were included. An inverse correlation between the peripheral frequencies of NK cells and Tregs was found in NA suppressed patients following pegylated interferon-alpha (PegIFN-alpha)-based treatment. Furthermore, NK cells suppressed the proliferation and differentiation of Tregs through secreting IFN-gamma as was evidenced in the circulation of NA-treated CHB patients as well as in liver of HBV-carrier mouse model. Additionally, the inhibition could be enhanced by PegIFN-alpha treatment, which was correlated to more vigorous HBV-specific T-cell responses and marked reduction in HBsAg. Our study reveals a novel immunomodulatory mechanism of NK cells and provides a theoretical basis for PegIFN-alpha as an immunotherapy agent in treating patients with CHB.