Gastro-oesophageal reflux disease (GERD) is a multi-factorial disease that may involve oesophageal hypersensitivity to mechanical or heat stimulus as well as acids. Intraganglionic laminar endings (IGLEs) are the most prominent terminal structures of oesophageal vagal mechanosensitive afferents and may modulate mechanotransduction via purinergic receptors. Transient receptor potential channel vanilloid 4 (TRPV4) can detect various stimuli such as warm temperature, stretch and some chemicals, including 4α-phorbol 12,13-didecanoate (4α-PDD) and GSK1016790A. TRPV4 is expressed in many tissues, including renal epithelium, skin keratinocytes and urinary bladder epithelium, but its expression and function in the oesophagus is poorly understood. Here, we show anatomical and functional TRPV4 expression in mouse oesophagus and its involvement in ATP release. Methods: Expression studies were performed on mouse esophagus by RT-PCR and immunostaining. TRPV4 channel activities were examined by Ca2+-imaging and Patch-Clamp experiments with primary esophageal keratinocytes isolated from WT and TRPV4KO mice. ATP release from keratinocytes was measured by Luciferin-Luciferase assay. Results: TRPV4 mRNA and protein were detected in esophageal keratinocytes. Several TRPV4 activators such as chemicals, heat and stretch stimulus have increased cytosolic Ca2+ concentrations in primary WT keratinocytes, but not in TRPV4KO keratinocytes. GSK and heat stimulus evoked TRPV4-like current responses in WT cells, but not in TRPV4KO cells. The mRNA of a newly identified ATP transporter, VNUT and its protein were also detected in esophageal WT keratinocytes. TRPV4 activators (GSK and heat stimulus) increased ATP release from WT cells, but not from TRPV4KO cells. This release was inhibited by a vesicle-trafficking inhibitor brefeldin A. Conclusion: TRPV4 contributes to ATP release via exocytosis, responding to heat, chemical and possibly mechanical stimuli. TRPV4 could be involved in esophageal mechano- and heat-hypersensitivity and be related to the pathophysiology of GERD.