Aquaporin-3 (AQP3) plays an important role in maintaining the normal water content of the skin. Previously, we revealed that the expression of cutaneous AQP3 increased following oral administration of Gypsum fibrosum (main component: CaSO4) to mice. The purpose of this study is to elucidate the mechanism by which Gypsum fibrosum increases the expression of cutaneous AQP3 in a keratinocyte cell line. Gypsum fibrosum or CaSO4 was added to keratinocytes, and the expression level of AQP3, the Ca concentration, the activity of protein kinase C (PKC), and the degrees of phosphorylation of both extracellular signal-regulated kinase (ERK) and cAMP response element binding protein (CREB) were measured. The mRNA and protein expression levels of AQP3 increased significantly 6 h-post addition of Gypsum fibrosum. In keratinocytes treated with Gypsum fibrosum, increases in the concentration of intracellular Ca, PKC activity, and the phosphorylation of ERK and CREB were observed. Pre-treatment with GF109203X, a PKC inhibitor, suppressed the mRNA expression levels of AQP3. Similarly to treatment with Gypsum fibrosum, the addition of CaSO4 led to the same observations in keratinocytes. It is hypothesized that Gypsum fibrosum causes an increase in the intracellular Ca concentration, PKC activity, and the phosphorylation levels of ERK and CREB, resulting in increased AQP3 expression in keratinocytes. In addition, it is possible that the effect of Gypsum fibrosum is attributable to CaSO4, based on the results demonstrating that the mechanisms of action of Gypsum fibrosum and CaSO4 were nearly identical.