TRPV4 is a cationic channel activated by hypotonicity, moderated heat and shear stress. In non-excitable cells it is involved in cellular functions such as volume regulation, migration and differentiation. Here, we studied how the expression of TRPV4 changes during proliferation and differentiation of the rabbit corneal epithelial cell model RCE1(5T5), (RCE1) and its role in differentiation. RCE1 is a spontaneously transformed cell line that recapitulates in vitro the early stages of differentiation. In early cultures, the cells are proliferative, and after 10-14 days, a stratified epithelium of 5-6 layers is formed (García-Villegas et al., 2009). TRPV4 expression was measured by qRT-PCR and Western Blotting. Subcellular localization of TRPV4 and TRPP2 was detected by immunofluorescence. Confocal Ca2+-imaging with Fluo-4AM was used to determine the TRPV4 activity together with the TRPV4 specific activator GSK1016790A (GSK101, 100 nM), the specific blocker RN-1734 (30 µM) or EGTA (2 mM) in sodium Ringer’s solution (in mM: NaCl 145, KCl 5, CaCl2 1, KH2PO4 1, MgCl2 1, glucose 10, HEPES 10, pH 7.4). Transepithelial electric resistance (TER) was measured with an EVOM voltmeter in cultures grown on 24-transwells inserts. All data are means ± SEM of at least 2 independent experiments in triplicate. TRPV4 mRNA levels remained unchanged along the proliferation and differentiation of RCE1 cells. Protein expression levels of TRPV4 in differentiated cultures decreased 70% and the channel was localized in the apical membrane of the outmost cell layer of stratified epithelia. TRPV4 channel is functional in differentiated cultures since the perfusion with GSK101 promoted an important influx of Ca2+ (2 ± 0.35 ΔF/F0, n=71), which was blunted by RN-1734 or EGTA. In our study, we analyzed the participation of TRPV4 in RCE1 differentiation by measuring the development of the TER as an indicator of tight junctions (TJ) assembly. Proliferative RCE1 cell cultures chronically treated with RN-1734 from day 7-14 exhibited only 66 % of the TER value (107 ±11 Ω●cm2) measured in control cultures (162 ± 5 Ω●cm2) and no effect was observed after GSK101 treatment. In differentiated epithelia, activation of TRPV4 increased TER by 30% (220 ± 8 Ω●cm2). EGF (10 ng/ml) up-regulates the TER of RCE1 cultures and here we also observed that TRPV4 activation mimics this EGF effect. Conversely, TRPV4 inhibition prevents the increase of TER even in the presence of the growth factor. TRPP2, an EGF-activated channel, co-localizes with TRPV4 in differentiated cultures suggesting that the EGF regulation of TJ may involve a heterotretrameric TRPV4-TRPP2 channel. Together our results demonstrate that TRPV4 activity is necessary for the correct establishment of TJ in corneal epithelia and also for regulating both the barrier function of TJ and its ability to respond to EGF.