Mifepristone (RU486) is developed originally as a contraceptive used by hundreds of millions of women world-wide, and also reported as a safe and long-term psychotic depressant, or as a cancer chemotherapeutic agent used by both sexes. In our preliminary study aimed at developing mifepristone as a cancer metastatic chemopreventive, we coincidentally observed that blood mifepristone concentrations in female rats seem to be higher than those in male ones post administration. To substantiate if the pharmacokinetic differences between sexes exist, we established a fast UPLC–MS/MS method to determine mifepristone concentrations in plasma, and analyzed blood concentrations of mifepristone over time in rats and dogs of both sexes. Mifepristone in plasma or incubation liquid was recovered by liquid-liquid extraction using 1mL of ethyl acetate. Chromatographic separation was performed on a C18 column at 35°C, with a gradient elution consisting of methanol and water containing 0.1% (v/v) formic acid at a flow rate of 0.3mL/min. And pharmacokinetic parameters such as elimination half-life, and mean residence time were calculated by using the non-compartmental pharmacokinetics data analysis software. In this work, administrations of mifepristone to rats and beagle dogs revealed that the plasma concentrations of mifepristone (AUC, Cmax) were significantly higher (P<0.05) in females than that in males. In vitro liver microsomal incubation experiments showed that the metabolic rate of mifepristone in males was higher than that in females, which was consistent with the results of in vivo experiments. In general, we first found the sex-related differences about pharmacokinetic properties of mifepristone and revealed the metabolism difference of hepatic microsomal enzyme is the main reason.