Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel that is widely expressed in different body tissues and plays several physiological roles. This channel is highly expressed in esophageal keratinocytes where its activation mediates ATP release. However, whether TRPV4 has a role in wound healing of esophageal keratinocytes is unclear. In this study, we demonstrated that both cell migration and proliferation were slower in wild-type esophageal keratinocytes compared to cells having TRPV4 knockout. Our results suggest that TRPV4-mediated release of ATP from esophageal keratinocytes contributes to a decrease in the rate of in vitro wound healing via the ATP degradation product adenosine, which acts on A2B adenosine receptors.