A UPLC-MS/MS method was developed to determinate curdione in the mouse blood, and the pharmacokinetics of curdione in mice after intravenous (5 mg kg−1) and oral (20 mg kg−1) administration were studied. The HSS T3 column was used for separation, and column temperature was set at 40 °C. Multiple reaction monitoring (MRM) mode were used for determination of curdione. Blood samples were taken from the caudal vein of Institute of Cancer Research (ICR) mice after administration of curdione. It showed a good linear relationship in the range of 1–500 ng mL−1 (r > 0.998); the intra-day precision was <13%, the inter-day precision was <15%, and the accuracy was 90%–105%, the recovery was >77%, and the matrix effect was 97%–107%. The half-life was relatively short, and the bioavailability was 6.5%. The developed method was suitable for the pharmacokinetics of curdione in mice.