Objective To explore the effects and the possible molecular mechanism of flavonoids of puerarin (PR) on chronic myelogenous leukemia (CML) cell line K562 and acute promyelocytic leukemia (APL) cell line NB4 in vitro. Methods MTT assays were used to detect the inhibitory effects of cell proliferation. The apoptosis of K562 and NB4 cells was detected by flow cytometry marked with Annexin V/PI. The expression of bcr-abl, p53, bcl-2, Fas/FasL in K562 cells and JNK, PARP, bcl-2 and Caspase 3 in NB4 cells at protein level was detected by Western blot. Results PR could inhibit the proliferation of K562 and NB4 cells in a time-dose dependent manner. The expression of protein levels of bcr-abl fusion gene declined, while the p53 protein otherwise increased, and both were in a dose-dependent manner (F = 18.74, P <0.05). The application of PR had no effect on bcl-2 and Fas/FasL protein expression in K562 cells. The JNK, PARP and Caspase3 proteins were upregulated in NB4 cells, while bcl-2 was downregulated with the increasing concentrations of PR (F=42.32, P <0.05). Conclusion PR could inhibit leukemic cell proliferation, induce cell cycle block, and increase cell apoptosis through different molecular mechanisms. It suggestes that PR might potentially be a kind of broad spectrum anti-leukemia agent.