The objective of this study was to determine factors associated with hepatitis B surface antigen (HBsAg) seroclearance after hepatitis B e-antigen (HBeAg) seroclearance.This is a cohort study of HBeAg-positive patients with HBeAg seroclearance. Factors associated with subsequent HBsAg seroclearance were examined.A total of 775 patients were included. At 1, 5, 10, 15, 20, and 25 years after HBeAg seroclearance, the HBsAg seroclearance rate was 0.3, 1.3, 3.0, 8.9, 15.7, and 23.6%, respectively. The rate of HBsAg seroclearance was highest in those who underwent spontaneous HBeAg seroclearance and required no treatment afterward (group 1), compared with those who underwent treatment-induced HBeAg seroclearance (group 2), and those who required antiviral therapy after spontaneous HBeAg seroclearance (group 3). At 25 years after HBeAg seroclearance, the HBsAg seroclearance rate was 38.0, 14.9, and 0% in groups 1, 2, and 3, respectively (P<0.001). There was no difference in the rate of HBsAg seroclearance between those who received interferon-based therapy compared with nucleos(t)ide analogs. The median HBV DNA level was similar between those with and without HBsAg seroclearance. The median HBsAg level was significantly lower in those who had HBsAg seroclearance compared with those who did not achieve loss of HBsAg (2.81 vs. 3.52 log IU/ml, respectively, P=0.009). The area under receiver operating characteristic curve for HBsAg at 1 year after HBeAg seroclearance for predicting HBsAg seroclearance was 0.742, with an optimal cutoff of 751 IU/ml.Spontaneous HBeAg seroclearance without need for subsequent antiviral therapy was associated with the highest rate of subsequent HBsAg seroclearance. Lower HBsAg levels were also associated with higher chance of HBsAg seroclearance.