Betaine (BET) is a native compound known for its ability to protect the liver from toxicants. However, few studies have examined the effects of BET on the most common cause of liver disease, hepatitis B virus (HBV). In this study, the anti-HBV activity of BET was assessed in vitro and in vivo using enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, and Southern blotting. The resistance of HBV to lamivudine and interferon alpha is challenging in the clinical treatment of HBV. The effect of BET on resistance was also investigated. The results showed that the secretion of HBsAg (HBV surface antigen), HbeAg (HBV e antigen), and HBV DNA in HepG2.2.15 cells was significantly decreased by BET via suppression of GRP78 expression. In duck HBV (DHBV)-infected ducklings, 1.0 or 2.0 g/kg BET significantly reduced serum DHBV DNA, and DHBV DNA did not rebound after the 5 day withdrawal period. BET suppressed HBV DNA rebound produced by the resistance of HBV to lamivudine and decreased the resistance mutation (rtM204V/I) of HBV DNA. Supplementation of BET may improve the anti-HBV effect of interferon alpha by increasing the expression of antiviral dsRNA-dependent protein kinase induced by the JAK-STAT (JAK = Janus kinase; STAT = signal transducer and activator of transcription) signaling pathway. These results may provide useful information for the clinical application of BET and solution of HBV drug resistance in anti-HBV therapy.