Major sex differences are observed in the prevalence, intensity, and severity of hepatitis B virus (HBV) infection. Here, we investigated degranulation activity of circulating and intrahepatic natural killer (NK) cells from HBV and HCV chronically infected patients before any treatment (n = 125). The frequency of CD107+ NK cells in the female liver was significantly higher compared to that in males during chronic HBV infection (p = 0.002) and correlated with the plasma levels of estradiol (correlation coefficient r = 0.634; p < 0.0001). Our results clearly show sex differences in degranulation activity of intrahepatic NK cells of HBV-infected patients. This probably contributes to the ability of females to better deal with HBV disease.