Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway is critical for the survival of differentiating cells and depends on the E-cadherin-catenin complex. In an earlier study we showed impaired PI3K/AKT activation in vitiliginous keratinocytes (KCs). Recently, aquaporin3 (AQP3) has been reported to co-accumulate with E-cadherin in forming cell-to-cell contacts. Therefore, we examined the expression of AQP3 in vitiliginous KCs and the role of AQP3 in KC survival and differentiation by comparing downstream signaling molecules. AQP3 protein expression was significantly decreased in the depigmented epidermis compared with the normally pigmented epidermis of patients with vitiligo. Transfection of cultured normal human KCs with AQP3 small interfering RNA (siRNA) reduced the expression levels of phosphorylated PI3K, E-cadherin, beta-catenin, and gamma-catenin, regardless of the calcium concentration. These downstream signaling molecules were also decreased in the depigmented epidermis. The results of immunoprecipitation and double staining confirmed colocalization of AQP3 with E-cadherin, as well as an active role of AQP3 in E-cadherin expression of cell-to-cell contacts. Moreover, AQP3 knockdown induced no increase in differentiating markers at high calcium concentrations and reduced survival of KCs, suggesting that reduced AQP3 in vitiliginous KCs might be responsible for their reduced survival.