The ability of entecavir (ETV) to inhibit Duck hepatitis B virus (DHBV) infection in duck hepatocytes and ducklings was examined using lamivudine (3TC) as a comparator drug. ETV exhibited antiviral activity (50% effective concentration [EC(50)], 0.13 nM) in DHBV-infected duck hepatocytes that was >1,000-fold more potent than that of 3TC (EC(50), 138 nM). A 21-day treatment of ducklings with 1 mg of ETV per kg of body weight per day by oral gavage resulted in a mean reduction of log(10) 3.1 in serum DHBV DNA levels. Daily treatment with 0.1 mg of ETV/kg was nearly as effective, achieving an average viral DNA level decrease of log(10) 2.1. Reducing the daily dose of ETV to only 0.01 mg/kg resulted in an average viral DNA level decrease of log(10) 0.97. Daily treatment with 25 mg of 3TC/kg resulted in an average viral DNA level decrease of log(10) 0.66, compared to the log(10) 0.20 drop seen for ducklings given the vehicle alone. ETV was also more effective in decreasing the DHBV DNA levels in duck livers after 21 days of treatment, causing average drops of log(10) 1.41, log(10) 0.76, and log(10) 0.26 for dose levels of 1.0, 0.1, and 0.01 mg/kg, respectively, compared to a decrease of log(10) 0.06 for 3TC at a dose level of 25 mg/kg. Levels of viral covalently closed circular DNA in the treatment group receiving 1 mg of ETV/kg were reduced compared to those in the vehicle-treated group. ETV and 3TC were both well tolerated in all treated animals. These results show that ETV is a highly potent and effective antiviral in the DHBV duck model.