The esophageal epithelium has sensory properties that enable it to sustain normal barrier function. Transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+)-permeable channel that is activated by extracellular hypotonicity, polyunsaturated fatty acids, phorbol esters, and elevated temperature. We found that TRPV4 is expressed in both human esophageal tissue and in HET-1A cells, a human esophageal epithelial cell line. Specific activation of TRPV4 by the phorbol ester 4α-phorbol 12,13-didecanoate (4α-PDD) increased intracellular Ca(2+) in a subset of HET-1A cells. Elevated temperature strongly potentiated this effect at low concentrations of 4α-PDD, and all of the responses were inhibited by the TRPV antagonist ruthenium red. TRPV4 activation differentially affected cell proliferation and cell viability; HET-1A cell proliferation was increased by 1 μM 4α-PDD, whereas higher concentrations (10 μM and 30 μM) significantly decreased cell viability. Transient TRPV4 activation triggered ATP release in a concentration-dependent manner via gap-junction hemichannels, including pannexin 1 and connexin 43. Furthermore, TRPV4 activation for 24 h did not increase the production of interleukin 8 (IL-8) but reduced IL-1β-induced IL-8 production. Small-interference RNA targeted to TRPV4 significantly attenuated all of the 4α-PDD-induced responses in HET-1A cells. Collectively, these findings suggest that TRPV4 is a novel regulator of Ca(2+)-dependent signaling pathways linked to cell proliferation, cell survival, ATP release, and IL-8 production in human esophageal epithelial cells.