It is thought that sensory nerve fibers contribute to the control of vasomotor tone by the release of calcitonin gene‐related peptide (CGRP). We stimulated the isolated rat arterial mesenteric bed with 1μM capsaicin or 100nM CGRP for 20 min and assessed the changes in nitric oxide (NO) signaling and hemichannel activity. Hemichannel activity was evaluated by ethidium or Lucifer Yellow (LY) uptake, expression and phosphorylation of eNOS by Western blot and NO‐dependent vasodilation with acetylcholine (ACh). Mesenteric arteries did not show detectable hemichannel activity in control conditions, but stimulation with either capsaicin or CGRP induced a robust ethidium or LY uptake that was maintained for >1 hr and was inhibited by probenecid, a pannexin hemichannel blocker. Capsaicin treatment induced a decrease in eNOS expression and eNOS phosphorylation at serine 1177 that reached significance 1–3 hrs after stimulation. Accordingly, the NO‐dependent ACh‐induced vasodilation was abolished 1 hr after capsaicin application, which was prevented by treatment with probenecid. These results suggest that CGRP release from capsaicin‐sensitive sensory nerves induces a prolonged activation of pannexin‐based hemichannels in endothelial cells, which leads to a reduction in NO signaling in mesenteric resistance arteries.FONDECYT 1100850, FONDECYT 1090757, FONDECYT 1111033, Anillos ACT‐71