This retrospective analysis was conducted to describe the characteristics of nucleoside-naïve hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B, who achieved hepatitis B surface antigen (HBsAg) loss during entecavir or lamivudine therapy. HBeAg-positive adults with chronic hepatitis B, elevated serum alanine aminotransferase, and compensated liver disease were randomized to double-blind treatment for up to 96 weeks with entecavir 0.5 mg/day or lamivudine 100 mg/day. HBsAg and hepatitis B virus (HBV) DNA were measured at regular intervals during and off-treatment follow-up. Through a maximum duration of 96 weeks on-treatment and 24 weeks off-treatment, HBsAg loss was confirmed in 18/354 (5.1%) patients treated with entecavir and 10/355 (2.8%) patients treated with lamivudine. Among the 28 patients with confirmed HBsAg loss, 27 (96%) achieved HBV DNA <300 copies/mL, and 27 (96%) achieved confirmed HBeAg loss. All entecavir recipients with HBsAg loss had HBV DNA <300 copies/mL. Caucasian patients, and those infected with HBV genotype A or D, were significantly more likely to lose HBsAg. This retrospective analysis of data from a randomized, global phase three trial shows that confirmed loss of HBsAg occurred in 5% of nucleoside-naïve HBeAg-positive patients treated with entecavir, and that HBsAg loss is associated with sustained off-treatment suppression of HBV DNA.