Endothelial cells express a variety of TRP (transient receptor potential channel) channels, including TRPV4, TRPM4 and several canonical TRPCs. Here we focus on the functional role of TRPV4 in endothelial cells by using mouse aorta endothelial cells (MAEC cells) derived from humanely killed wild type (TRPV4+/+) mice and from TRPV4 knockout mice (TRPV4-/-). TRPV4 integrates a large variety of stimuli ranging from hypotonic cell swelling (HTS), temperature, and ligands e.g. 4α-phorbol 12,13-didecanoate (4α-PDD), to endogenous agonists such as arachidonic acid (AA), anadamide and epoxyeicosatrienoic acids. We show that TRPV4 is likely a target for activation of endothelium-dependent vasorelaxation, which can be modulated via the cytochrome P450 (CYP) pathway. The loss of TRPV4 expression in MAEC cells from TRPV4-/- mice attenuated or strongly diminished responses to all TRPV4 activating stimuli. Ca2+ imaging and patch clamp measurements show that TRPV4-dependent responses can be modulated via CYP enzymes, which metabolize AA to EETs. MAEC from TRPV4+/+ mice preincubated with nifedipine, which upregulates CYP2C expression, show a potentiated response to AA and cell swelling, whereas activation by 4α-PDD. Sulfaphenazole, an inhibitor of CYP2C9, decreased responses induced by AA and HTS. 1-Adamantyl-3-cyclo-hexylurea (ACU), an inhibitor of the soluble epoxide hydrolase which converts EETs to dihydroxyeicosatrienoic acids, increased the response induced by AA, HTS and EETs but not that induced by 4α-PDD and heat. All these data demonstrate that cytochrome P450-derived EETs modulate the activity of TRPV4 channels in endothelial cells.