Liver x receptors (LXRs) play important role in the regulation of cholesterol and glucose homeostasis by regulating several transporters. In this study, we investigated the effects of LXRs on OAT1‐mediated organic anion transport in renal S2 cells expressing hOAT1 and mouse kidney. Exposure the S2 cells to LXRs agonists (TO901317 and GW3965) and their endogenous ligand (22(R)‐hydroxycholesterol) decreased OAT1‐mediated [14C]‐PAH uptake. This inhibition was abolished by co‐incubation with 22(S)‐hydroxycholesterol, a LXRs antagonist. Kinetic analysis revealed that LXRs activation decreased the maximum rate of PAH transport (Jmax) but had no effect on the affinity of the transporter (Kt). Data from western blot analysis showed the decrease in hOAT1 protein expression following LXRs activation. Similarly, we found the inhibition of [14C] PAH uptake in renal cortical slices as well as a decreased mOAT1 protein expression in mouse kidney. Our findings indicated for the first time that OAT1 function was downregulated by LXRs activation in renal proximal tubule.This work is supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program.