Background: Long-term treatment of chronic hepatitis B (CHB) with nucleos(t)ide analogs results in the emergence of drug-resistant hepatitis B virus (HBV) harboring mutations in the polymerase (P) gene. The Phyllanthus extract has anti-HBV activity; however, its antiviral activity against lamivudine (LMV)-resistant mutants has not been examined. Methods: HBV harboring LMV-resistant mutations (rtM204I, rtM204V, and rtM204S) in the P gene at the YMDD ((203)tyrosine-methionine-aspartate-aspartate(206)) reverse transcriptase (RT) active site were generated and their sensitivity to Phyllanthus urinaria koreanis extract examined. Southern blotting and real-time PCR were used to determine the concentration of plant extract required to inhibit HBV DNA synthesis by 50 and 90 % (EC50 and EC90, respectively). An enzyme-linked immunosorbent assay was used to measure the EC50 of HBV surface antigen (HBsAg) and HBV core antigen (HBcAg) secretion, and the 50 % cytotoxic concentration of the extract was measured in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Real-time RT-PCR was used to measure mRNA expression levels. Results: The expression of intracellular HBV DNAs in HBV WT- or mutant-transfected HepG2 cells decreased upon treatment with Phyllanthus extract. The secretion of HBsAg and HBcAg also fell in a dose-dependent manner. Phyllanthus extract induced interferon-beta (IFN-beta), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) mRNA expression in HBV WT-transfected HepG2 cells, possibly via activation of extracellular signal-regulated kinases and c-jun N-terminal kinases and the induction of retinoic acid inducible gene-I, toll-like receptor 3, myeloid differentiation primary response gene 88, and/or tumor necrosis factor receptor-associated factor 6 gene expression. HBV transfection in the absence of extract or exposure of cells to extract alone did not trigger these signaling cascades. Conclusions: Phyllanthus extract inhibited HBV DNA synthesis and HBsAg and HBcAg secretion by replicating cells harboring HBV wild-type and LMV-resistant mutants, likely by inducing the expression of IFN-beta, COX-2, and IL-6. These data indicate that Phyllanthus extract may be useful as an alternative therapeutic agent for the treatment of drug-resistant CHB patients.