AIM: To investigate the safety of β-L-D4A on DNA polymerase α.METHODS: Ion exchange chromatography was used to separate DNA polymerase α from crude extract of human Hela cells. Detailed kinetic parameters were determined for β-L-D4A against DNA polymerase α.RESULTS: DNA polymerase α was purified with 4% yield and 31000 units/mg specific activity. The Michaelis constant (Km = 3.22 μmol/L), 50% inhibition concentration (IC50 = 178.49 μmol/L) and inhibition constant (Ki = 126 μmol/L) of β-L-D4A were determined by kinetic analysis.CONCLUSION: β-L-D4A is a more safe nucleoside for hepatitis B virus (HBV) infection with a lower host toxicity.