Local calcium (Ca2+) signals are crucial for endothelial functions, including regulation of underlying smooth muscle cell contractility. Ca2+ pulsar is an endothelial IP3R‐dependent Ca2+ signal restricted to myoendothelial projections (MEP). Targets for this spatially‐discrete Ca2+ signal such as KCa3.1 channels, NOS3 and CaMKII have been previously described. However, determinants for Ca2+ pulsar kinetics and stochasticity remains poorly understood. Mitochondria are an important regulator of intracellular Ca2+ dynamics in several cell types such as pancreatic acinar cells. It has thus been hypothesized that mitochondria are an important modulator of Ca2+ pulsar. Mitochondrial influence on Ca2+ pulsar was explored on cut‐opened mesenteric resistance arteries using a high‐speed confocal microscope. Inhibition of mitochondrial uniporter‐dependent Ca2+ uptake (Ru360) increased the surface covered by a Ca2+ pulsar (+30%). Conversely, stimulation of mitochondrial Ca2+ uptake (kaempferol) reduced Ca2+ pulsar's spreading (−20%), suggesting that mitochondria could limit Ca2+ pulsars propagation. Interestingly, inhibition of mitochondrial Ca2+ efflux (CsA for mPTP; CGP37157 for NCLX) had little if any effect on spatial dispersion of a Ca2+ pulsar. However, de novo Ca2+ pulsar sites were observed following exposure to CsA/CGP. Mitochondria might thus control subcellular Ca2+ levels within MEP to modulate IP3R activity. Electron microscopy showed clustering of mitochondrial distribution near the MEP. These results suggest that mitochondria might play a significant role in regulating local endothelial Ca2+ signalling and then, modulate vascular tone.Support or Funding InformationFRQS, CFI, SQHA and CIHR