BACKGROUND Celastrol is a natural compound extracted from the traditional Chinese medicinal herb, Thunder God Vine (TGV). Owing to its potential anti-inflammatory and antitumor effects, celastrol has been considered as a promising candidate for drug development. OBJECTIVE To establish a sensitive LC-MS/MS method to investigate the pharmacokinetic properties of celastrol in rats. Key pharmacokinetic issues of celastrol including oral bioavailability, comparative pharmacokinetics between pure compound and tablet preparation, as well as gender-related pharmacokinetic difference are to be addressed for the first time. METHODS Sprague-Dawley rats were administrated an intravenous dose (100 μg kg(-1)) of pure celastrol and an oral dose (1000 μg kg(-1)) of pure celastrol and TGV tablets (corresponding to 534 μg kg(-1) of celastrol), respectively. At different time points, the concentration of celastrol in rat plasma was determined by a sensitive and well-validated LC-MS/MS method. Main pharmacokinetic parameters including area under the plasma concentration-time curve (AUC), maximal plasma concentration (Cmax), the time for maximal concentration (Tmax) and mean residence time (MRT) were estimated by Drug and Statistic1.0 pharmacokinetic software (Chinese Pharmacological Association, Anhui, PR China). Statistical analysis was performed using two one-side t test with p-values less than 0.05 as the level of significance. RESULTS The standard curve of celastrol showed good linearity in the concentration range of 0.11~54.3 ng mL(-1) in our current method, with acceptable selectivity, precision, recovery, and stability. The oral absolute bioavailability of celastrol significantly increased from 17.06% for pure celastrol to 94.19% for TGV tablets containing equivalent celastrol. After oral administration of TGV tablets, the Cmax and AUC values of celastrol in female rats were (32.03±8.41) μg L(-1) and (379.49±118.19) μg h L(-1), which were significantly higher (p<0.01) than that in males with the values of (14.31±7.33) μg L(-1) and (188.17±92.33) μg h L(-1). CONCLUSIONS Celastrol administered orally in the rat was poorly absorbed into the systemic circulation. However, the poor absorption of celastrol could be greatly improved when celastrol-containing TGV tablets orally administered, and thereby the oral bioavailability of celastrol was significantly increased. As for gender difference, female rats showed significantly better absorption of celastrol than males.