Endothelial cell (EC) Ca²⁺-activated K channels (SK_Ca and IK_Ca channels) generate hyperpolarization that passes to the adjacent smooth muscle cells causing vasodilation. IK_Ca channels focused within EC projections toward the smooth muscle cells are activated by spontaneous Ca²⁺ events (Ca²⁺ puffs/pulsars). We now show that transient receptor potential, vanilloid 4 channels (TRPV4 channels) also cluster within this microdomain and are selectively activated at low intravascular pressure. In arterioles pressurized to 80 mmHg, ECs generated low-frequency (∼2 min⁻¹) inositol 1,4,5-trisphosphate receptor-based Ca²⁺ events. Decreasing intraluminal pressure below 50 mmHg increased the frequency of EC Ca²⁺ events twofold to threefold, an effect blocked with the TRPV4 antagonist RN1734. These discrete events represent both TRPV4-sparklet- and nonsparklet-evoked Ca²⁺ increases, which on occasion led to intracellular Ca²⁺ waves. The concurrent vasodilation associated with increases in Ca²⁺ event frequency was inhibited, and basal myogenic tone was increased, by either RN1734 or TRAM-34 (IK_Ca channel blocker), but not by apamin (SK_Ca channel blocker). These data show that intraluminal pressure influences an endothelial microdomain inversely to alter Ca²⁺ event frequency; at low pressures the consequence is activation of EC IK_Ca channels and vasodilation, reducing the myogenic tone that underpins tissue blood-flow autoregulation.