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The aim of the present study was to discover human mitotic kinesin protein Eg5 inhibitors.The human Eg5 eDNA was obtained by using RT-PCR method with total RNA extracted from the HL60 cells,then was cloned into the expression vector pET28a and transformed into Escherichia coli BL21 (DE3).The protein was produced by IPTG induction,and then purified in a two-step process.A molecular level in vitro screening assay for Eg5 inhibitors was set up to screen a set of 300 pure compounds in a small-moleenlar library.Then a cell-based MTT assay was used to assess the biological activity of these compounds in terms of human tumor cells growth and death.Immunofluoresoence staining of β-miorotubules and DNA was used to verify the mechanism.A DNA fragment encoding N-terminal Eg5 protein (Swissprot P52732) was amplified by PCR and sub-cloned into the pET28a.The molecular weight of expressed product was in good agreement with what the SDS-PAGE analysis showed.12 potential "hits" were identified from this small-molecular library.They appeared more potent than monastrol,little less potent than CK0106023.They could induce a distinct diminution of human tumor cells viability.CPUYL001 and CPUYL064 caused the monastrol spindle instead of the normal bipolar spindle in the mitotic stage of the cells according to the results of immunofluorescence staining assay.The specific inhibitors of Eg5 may have clinical utility in the treatment of cancer in the future.