Curcumin (CUR) is known to exert numerous health-promoting effects in pharmacological studies, but its low bioavailability hinders the development of curcumin as a feasible therapeutic agent. Piperine (PIP) has been reported to enhance the bioavailability of curcumin, but the underlying mechanism remains poorly understood. In an attempt to find the mechanism by which piperine enhances the bioavailability of curcumin, the dosage ratio (CUR: PIP) and pre-treatment with piperine were hypothesized as key factors for improving the bioavailability in this combination. Therefore, combining curcumin with piperine at various dose ratios (1:1 to 100:1) and pre-dosing with piperine (0.5-8h prior to curcumin) were designed to investigate their contributions to the pharmacokinetic parameters of curcumin in rats and their effects on the expression of UGT and SULT isoforms. It was shown that the C-max and AUC(0-t) of curcumin were slightly increased by 1.29 and 1.67 fold at a ratio of 20:1, while curcumin exposure was enhanced significantly in all the piperine pre-treated rats (0.5-8h), peaking at 6h (a 6.09-fold and 5.97-fold increase in C-max and AUC(0-t), p<0.01), regardless of the unchanged t(1/2) and T-max. Also observed was a time-dependent inhibition of the hepatic expression of UGT1A6, 1A8, SULT1A1, 1A3, and the colonic expression of UGT1A6 that occurred within 6h of piperine pre-treatment but was reversed at 8h, which correlated with the changes in curcumin exposure. Similarly, the inhibitory effect of piperine on most of the UGTs and SULTs are time-dependent in Caco-2 and HepG2 cells. It is concluded that piperine pre-treatment time-dependently improves the bioavailability of curcumin through the reversible and selective inhibition of UGTs and SULTs. Copyright (c) 2016 John Wiley & Sons, Ltd.