Aim Eg5 is a mitotic kinesin that plays a crucial role in the formation of bipolar mitotic spindles, by hydrolyzing ATP to push apart anti-parallel microtubules.Dimethylenastron is potent specific small molecule inhibitor of Eg5.The mechanism by which dimethylenastron inhibits Eg5 function remains unclear.In this study, by comparing with the analogue enastron,we sought to determine the molecular basis for the potent Eg5 inhibition by dimethylenastron.