Abstract Ethnopharmacological relevance Rhein is a pharmacological active component found in Rheum palmatum L. that is the major herb of the San-Huang-Xie-Xin-Tang (SHXXT), a medicinal herbal product used as a remedy for constipation. Here we have investigated the comparative pharmacokinetics of rhein in normal and constipated rats. Microarray analysis was used to explore whether drug-metabolizing genes will be altered after SHXXT treatment. Materials and methods The comparative pharmacokinetics of rhein in normal and loperamide-induced constipated rats was studied by liquid chromatography with electrospray ionization tandem mass spectrometry (LC-MS/MS). Gene expression profiling in drug-metabolizing genes after SHXXT treatment was investigated by microarray analysis and real-time polymerase chain reaction (RT-PCR). Results A validated LC-MS/MS method was applied to investigate the comparative pharmacokinetics of rhein in normal and loperamide-induced constipated rats. The pharmacokinetic results demonstrate that the loperamide-induced constipation reduced the absorption of rhein. Cmax significantly reduced by 2.5-fold, the AUC decreased by 27.8%; however, the elimination half-life (t1/2) was prolonged by 1.6-fold. Tmax and mean residence time (MRT) were significantly prolonged by 2.8-fold, and 1.7-fold, respectively. The volume of distribution (Vss) increased by 2.2-fold. The data of microarray analysis on gene expression indicate that five drug-metabolizing genes, including Cyp7a1, Cyp2c6, Ces2e, Atp1b1, and Slc7a2 were significantly altered by the SHXXT (0.5g/kg) treatment. Conclusion The loperamide-induced constipation reduced the absorption of rhein. Since among the 25,338 genes analyzed, there were five genes significantly altered by SHXXT treatment. Thus, information on minor drug-metabolizing genes altered by SHXXT treatment indicates that SHXXT is relatively safe for clinical application.