The enantioseparation of four stereoisomers of palonosetron hydrochloride by micellar electrokinetic chromatography using sodium cholate as chiral surfactant was described. Sodium cholate was shown to be effective in separating palonosetron hydrochloride stereoisomers. For method optimization, several parameters such as sodium cholate concentration, buffer pH and concentration, the types and concentration of organic modifiers and applied voltage, on the enantioseparation were evaluated and the optimum conditions were obtained as follows: 30mM borate buffer (pH 9.40) containing 70mM sodium cholate and 20% (v/v) methanol with an applied voltage of 20kV. Under these conditions, baseline separation of palonosetron hydrochloride stereoisomers was achieved within 18min.