To investigate the stereoselective pharmacokinetics of itraconazole enantiomers in rats, four cis-ITR stereoisomers at the dose of 15 mg/kg were administered orally to rats. Blood was collected and single stereoisomer of ITZ and hydroxy-itraconazole(OH-ITZ)were determinated simultaneously by LC-MS/MS. Samples were extracted by protein precipitation with acetonitrile. Durashell HILIC column(100 mm×2. 1 mm, 5. 0 m)was used as the analytical column, while a mixture of solvent A(0. 02% acetic acid and 5 mmol/L ammonium acetate in water)and B(50% acetonitrile and 50% methyl alcohol)was used as the mobile phase. A 5. 5 min binary gradient elution(delivered at 0. 5 mL/min)was performed for the separation. LC-MS/MS was performed in positive ion mode with multiple reactions monitoring(MRM). The pharmacokinetic parameters of itraconazole enantiomers after the administration were estimated as follows: the plasma levels and AUC0-∞ of OH-(2S, 4R, 2R)-ITZ and OH-(2S, 4R, 2S)-ITZ were higher than those of OH-(2R, 4S, 2R)-ITZ and OH-(2R, 4S, 2S)-ITZ(P< 0. 001). At the same time, female rats exhibited greater cmax, t1/2, AUC0-∞ than male rats, and the absorption of male rats was more rapid than those of females. The findings indicate significant stereoselective differences in the pharmacokinetic parameters of itraconazole enantiomers and gender difference in rats.