Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily that is highly expressed in enterohepatic tissue, is implicated in bile acid, lipid, and glucose metabolisms. Although recent studies showed that FXR is also expressed in vascular endothelial cells and smooth muscle cells, its physiological and/or pathological roles in vasculature tissue remain unknown. The aim of this study is to examine the chronic effect of synthetic FXR agonist, GW4064, on vascular contraction and endothelium-dependent relaxation using tissue culture procedure. In cultured rabbit mesenteric arteries, the treatment with 0.1-10 µM GW4064 for 7 days did not influence vascular contractility induced by high K⁺ (15-65 mM), norepinephrine (0.1-100 µM) and endothelin-1 (0.1-100 nM). However, the chronic treatment with GW4064 (1-10 µM for 7 days) dose-dependently impaired endothelium-dependent relaxation induced by substance P (0.1-30 nM). In hematoxylin-eosin cross-sectioning and en face immunostaining, GW4064 had no effects on morphology of endothelial and smooth muscle cells. In endothelium-denuded arteries treated with GW4064 (1-10 µM) for 7 days, 3 nM-100 µM sodium nitroprusside-induced vasorelaxation, but not membrane permeable cGMP analog, 8-bromoguanosine-3',5'-cyclomonophosphate (8-Br-cGMP) (1-100 µM)-induced vasorelaxation was significantly impaired. In these GW4064-treated arteries, 1 µM sodium nitroprusside-induced intracellular cGMP elevations were impaired. In RT-PCR, any changes were detected in mRNA expression level of 1 and 1 subunits of soluble guanylyl cyclase (sGC). These results suggest that chronic stimulation of FXR impairs endothelium-dependent relaxation, which is due to decreased sensitivity of smooth muscle cells to nitric oxide.