Ca2+-influx through endothelial TRP channels has been proposed to play a significant role in the Ca2+ dependent production of vasodilators. Here, we investigated the role of the mechanosensitive TRPV4 in rat endothelial cells (EC) by using the in-situ patch-clamp technique, Ca2+-imaging and pressure-myography in carotid artery (CA) and A. gracilis. In EC, TRPV4-currents were activated by the TRPV4-opener 4aPDD, arachidonic acid, warmth and by hypotonic cell swelling. Activation of TRPV4 by 4aPDD increased [Ca2+]i by ≈140nM. In CA and A. gracilis, 4aPDD caused robust vasodilations (KD 0.3 mM) which were suppressed by the TRPV4-inhibitor ruthenium red (RuR). Wall shear stress (WSS)-induced vasodilation was similarly blocked by RuR and also by PLA2 inhibition. 4aPDD-induced vasodilation was mediated by NO and EDHF. WSS-induced vasodilations were mediated exclusively by NO. In conclusion, Ca2+-entry through endothelial TRPV4 triggers NO- and EDHF-dependent vasodilation. Moreover, TRPV4 appears to be mechanistically important in endothelial mechanosensing of WSS.