Hepatitis B virus (HBV) infection is a major public health problem worldwide. The study aimed to evaluate the efficacy of pegylated interferon (Peg-IFN) alfa-2a treatment for seroclearance of HBs antigen (HBsAg) in HBe antigen (HBeAg)-negative chronic hepatitis B (CHB) patients. This retrospective study investigated 16 HBeAg-negative CHB patients who received Peg-IFN alfa-2a weekly for 48weeks. Thereafter, the patients were followed-up for 48weeks after the end of therapy. The following criteria were also used for inclusion: HBV-DNA < 5.0 log copies/mL and without nucleot(s)ide analogs. Four HBsAg-positive cases became HBsAg negative. The HBsAg levels of the 4 patients who achieved HBsAg seroclearance were lower significantly than that of the non-seroclearance group (p = 0.007). The mean HBsAg levels in these 4 cases were 68IU/mL, while the mean HBsAg levels in the non-seroclearance group were 2,114IU/mL. The mean HBV-DNA levels in the 4 HBsAg seroclearance cases were 2.8 log copies/mL as compared to 3.6 log copies/mL in HBsAg-non-seroclearance cases (p = 0.01). Cases that are HBeAg negative, with HBV-DNA levels < 5 log copies/mL, and HBsAg titers < 120IU/mL cases may achieve HBsAg clearance with Peg-IFN therapy.