Background and Purpose Ca²⁺ influx via TRPV4 triggers Ca²⁺ release from the IP₃-sensitive internal store to generate repetitive oscillations. While mitochondria are acknowledged regulators of IP₃-mediated Ca²⁺ release, how TRPV4-mediated Ca²⁺ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca²⁺-induced Ca²⁺ release at IP₃ receptors, to being independent of Ca²⁺ influx and instead mediated by ATP release via pannexins. Experimental Approach TRPV4 evoked Ca²⁺ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520. Key ResultsTRPV4 activation with GSK1016790A(GSK) generated repetitive Ca²⁺ oscillations that required Ca²⁺ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca²⁺ waves in the absence of external Ca²⁺. The ATP synthase inhibitor oligomycin did not potentiate TRPV4 mediated Ca²⁺ signals. GSK-evoked Ca²⁺ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the phospholipase C (PLC) blocker U73122 and the inositol triphosphate receptor (IP₃ R) blocker caffeine. The Ca²⁺ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid. Conclusion and Implications These results highlight a previously unknown role of mitochondria in shaping TRPV4 mediated Ca²⁺ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4-mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP₃ evoked Ca²⁺ release.