--- - |2+ Abstract Aim Our earlier studies showed that mechanical stretch activates inhibitory motor neurons of the esophagus; however, the underlying molecular mechanisms are unclear. Here, we sought to examine if Na+/Ca2+ exchanger 1 (NCX1) is responsible for the mechanosensitivity in the esophageal myenteric neurons (EMN) of rats and humans. Methods The function of NCX1 in primary culture of neurons was determined using calcium imaging, and mechanosensitivity was tested using osmotic stretch and direct mechanical stretch. Axial stretch‐induced relaxation of the lower esophageal sphincter (LES) was also studied in vivo in rats. Results The expression and co‐localization of NCX1 with nNOS were identified in the EMN from both rats and humans. The extracellular Ca2+ entry caused by ATP through purinergic signaling in the rat EMN was significantly inhibited by selective NCX blockers. Removal of extracellular Na+ to activate the Ca2+ entry mode of NCX1 induced an increase in the cytoplasmic calcium ([Ca2+]cyt), which was attenuated by NCX blockers. Osmotic stretch and mechanical stretch‐induced [Ca2+]cyt signaling in the rat and human EMN were attenuated by NCX blockers as well as specific NCX1 knockdown. Osmotic stretch and mechanical stretch also induced [Ca2+]cyt signaling in the Chinese hamster ovary (CHO) cells with NCX1 over‐expression, which was attenuated by NCX blockers. Finally, NCX blockade inhibited axial stretch‐activated LES relaxation in‐vivo experiments in the rats. Conclusions We demonstrate a novel NCX1/Ca2+ pathway in the mechanosensitive neurons of rat and human esophagus, which may provide a potential therapeutic target for the treatment of esophageal motility disorders. This article is protected by copyright. All rights reserved. - 'Acta Physiologica, Volume 0, Issue ja, -Not available-. '