Curcumin (CUR), derived from dietary spice turmeric, is a polyphenolic compound with various biological and pharmacological activities. Tetrahydrocurcumin (THC) is one of the major reductive metabolites of CUR. A pharmacokinetic study using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for simultaneous determination of CUR, THC, quercetin (QR), and paeoniflorin (PF) in rat plasma had been performed. In this study, regional distributions of CUR and THC in the liver and the three segments of small intestine (duodenum, jejunum and ileum) of rats when orally co-administered with QR and PF were carried out. Drug concentrations were determined using UHPLC-MS/MS. The results showed that, CUR was well distributed in the small intestine, while the distributions of THC in the liver, duodenum, jejunum were similar, but much more abundant in the ileum. When orally co-administered with QR and PF, the tissue to plasma concentration ratios (Kp values) of CUR in the three segments of small intestine were increased, indicating that the presence of QR and PF increases the distribution of CUR in these regions. Moreover, the half-life (t1/2 ) of THC in the liver was significantly prolonged, and the Kp value of THC in the liver was increased and the Kp values in the small intestine were decreased, suggesting that the combination of QR and PF might suppress the metabolism of CUR in the small intestine. In brief, the combination had an effect on the distributions of CUR and THC in the liver and small intestine of rats. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.