Abstract: Objective To study the effect of rhein, a hepatitis B virus(HBV)reverse transcriptase(HBV-RT)inhibitor, combined with lamivudine on the activity of HBV-RT.Methods HBV-RT inhibitory activity and inhibition kinetic constants were studied with enzyme inhibition kinetics methods and the synergistic effect of rhein and lamivudine on HBV DNA replication was analyzed.Results The results showed that rhein was a reversible noncompetitive inhibitor of HBV-RT, with a 50% inhibitory concentration(IC₅₀)of 39.88 μmol/L and an inhibition constant Ki of 0.122 μmol/L.Circular dichroism spectra analysis showed that the binding of rhein to HBV-RT induced the unfolding of HBV-RT conformation, with a gradual increase of α-helix content(from 16.4% to 25.9%)and a gradual decrease of β-sheet(from 37.1% to 28.8%)of HBV-RT.Low concentration of heptodin combined with rhein exerted a synergistic effect on HBV DNA replication.Conclusion Rhein could reduce the activity of HBV-RT and affect HBV DNA replication synergistically when combined with heptodin at low concentration.