Objective To explore the effects of isobavachalcone (IBC) on imatinib-sensitive and imatinib-resistant chronic myelocytic leukemia cells. Methods Imatinib-sensitive chronic myelocytic leukemia cells K562s and imatinib-resistant chronic myelocytic leukemia cells K562r were cultured in vitro. K562s and K562r cells were treated by IBC of different concentrations (0, 5, 10, 20, and 40 μmol/L) for different period of time (0, 24, 48, and 72 h). The effects of IBC on the viability of K562s and K562r cells were detected by the trypan blue exclusion assay. The variations of cell apoptosis and mitochondrial membrane potential were detected by the Annexin V/PI staining and Rh123/PI staining, respectively. The expressions of proteins relevant to apoptosis were detected by the Western blotting. Results The results of trypan blue exclusion assay showed that IBC had an inhibitory effect on the proliferation of K562s and K562r cells and the effect was time and dose dependent. The results of flow cytometry indicated that the apoptosis and decrease of mitochondrial membrane potential of K562s and K562r cells treated by IBC were significantly time and dose dependent. The results of Western blotting showed that IBC induced activation of caspase-3 and cleavage of PARP-1 in K562s and K562r cells. Conclusion IBC can induce the apoptosis of K562s and K562r cells and the decrease of mitochondrial transmembrane potential may involve in this process.