Agonist-induced Ca²⁺ entry is important for the synthesis and release of vasoactive factors in endothelial cells. The transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca²⁺-permeant cation channel, is expressed in endothelial cells and involved in the regulation of vascular tone. Here we investigated the role of TRPV4 channels in acetylcholine-induced vasodilation in vitro and in vivo using the TRPV4 knockout mouse model. The expression of TRPV4 mRNA and protein was detected in both conduit and resistance arteries from wild-type mice. In small mesenteric arteries from wild-type mice, the TRPV4 activator 4α-phorbol-12,13-didecanoate increased endothelial [Ca²⁺]_i in situ, which was reversed by the TRPV4 blocker ruthenium red. In wild-type animals, acetylcholine dilated small mesenteric arteries that involved both NO and endothelium-derived hyperpolarizing factors. In TRPV4-deficient mice, the NO component of the relaxation was attenuated and the endothelium-derived hyperpolarizing factor component was largely eliminated. Compared with their wild-type littermates, TRPV4-deficient mice demonstrated a blunted endothelial Ca²⁺ response to acetylcholine in mesenteric arteries and reduced NO release in carotid arteries. Acetylcholine (5 mg/kg, IV) decreased blood pressure by 37.0±6.2 mm Hg in wild-type animals but only 16.6±2.7 mm Hg in knockout mice. We conclude that acetylcholine-induced endothelium-dependent vasodilation is reduced both in vitro and in vivo in TRPV4 knockout mice. These findings may provide novel insight into mechanisms of Ca²⁺ entry evoked by chemical agonists in endothelial cells.