Background and PurposeCa2+influx via TRPV4 channels triggers Ca2+release from the IP3‐sensitive internal store to generate repetitive oscillations. Although 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 IP3receptors to being independent of Ca2+influx and instead mediated by ATP release via pannexins.Experimental ApproachTRPV4‐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 PLC blocker U73122 and the inositol trisphosphate receptor blocker caffeine. The Ca2+waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid.Conclusion and ImplicationsThese 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.