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Objective PHⅡ-7 was a novel anti-cancer drug and was designed according to indirubin which was the active agent of traditional Chinese herb Danggui Longhui Wan.PH Ⅱ-7 was revealed to be a novel multifunctional drug which could not only inhibit proliferation of cancer cells, induce cell cycle arrest and apoptosis of cancer cells but also overcome multi-drug resistance by decreasing expression of multi-drug resistance gene MDR1 in multi-drug resistance cancer cells.Our aim was to evaluate the effects of PH Ⅱ-7 in chronic myeloid leukemia cell k562 and the effects of ENO1 which was one of the targets of PH Ⅱ-7 in chronic myeloid leukemia cell k562.Methods In our studies, stable ENO1 interfering chronic myeloid leukemia cell k562-shENO1 cell and control cell k562-shCON cell were screened by hygromycin.We used MTT assay to analyse the cytotoxicity and proliferation of chronic myeloid leukemia cell k562.Cell growth was assayed by counting cell numbers after phenol blue dyeing.Cell cycle and apoptosis was assayed by flow cytometer.And the expressions of relative genes on RNA level were assayed by realtime PCR and expressions of proteins were assayed by western blot.All experiments were repeated three times.Results Chronic myeloid leukemia cell k562-shENO1 cell and control cell k562-shCON cell were identified by realtime PCR and western blot, and results indicated that both interfering cells were valid.PH Ⅱ-7 inhibited the proliferation of k562-shENO1 cell and k562-shCON cell, IC50 was 0.98 ± 0.07μM and 0.89 ± 0.04 μM after being treated with PH Ⅱ-7 for 48h separately.PH Ⅱ-7 significantly inhibited cell growth in k562-shENO1 cell and k562-shCON cell, and the cell growths between k562-shENO1cell and k562-shCON cell was no different in vitro.Cell cycle was arrested in G2-M phrase in k562-shENO1 cell comparing with k562-shCON cell, and the expression of cyclinE was increased.PH Ⅱ-7 could induce apoptosis by activating some apoptosis relative proteins.Western blot revealed that the expressions of cl-PARP, cl-caspase3, cl-caspase9 and Bax were increased in k562-shENO1cell and k562-shCON cell, and k562-shENO1 cell was more sensitive to higher concentration of PH Ⅱ-7.Conclusion Our results highlighted the potential role of PHⅡ-7 in chronic myeloid leukemia cell k562.These findings revealed that PHⅡ-7 was a multi-functional anti-cancer drug, and it could regulate the expression of ENO1 which was proved to interact with PH Ⅱ-7 in vitro.ENO1 over-expression was associated with not only the cancer progression but also the poor clinical outcomes, and it could be considered as a novel target for inhibiting the tumorigenesis and overcoming drug resistance.PH Ⅱ-7 could influence the expressions of cell cycle relative genes and apoptosis relative genes through regulating the expression of ENO1.Taken together, ENO1, is an important target of PH Ⅱ-7 in chronic myeloid leukemia cell k562 and is related to cell cycle and apoptosis.