Desmoglein-3 (DSG3) is a critical molecule for adhesion between keratinocytes. Its expression can affect interactions between keratinocytes as well as keratinocyte morphology. Vitamin D3 (VD3) has been known for its role in mineral homeostasis, but has now been shown to be involved in cell differentiation, cell proliferation, immune responses and inflammation as well. Topical application of VD3 is used for the treatment of various skin diseases in present clinical practice. This study examined the effect of VD3 on DSG3 gene expression in keratinocytes. Incubation of the cultured keratinocytes with VD3 resulted in a significant decrease in Dsg3 mRNA expression. Cycloheximide treatment did not alter the inhibition rate of Dsg3 gene expression by VD3. We additionally measured the effect of VD3 on the promoter activity of the DSG3 gene. VD3 failed to inhibit promoter activities. These results suggest that down-regulation of Dsg3 expression by VD3 is independent of active protein synthesis, and may also result from keratinocyte regulatory mechanisms operating at the post-transcriptional level. Thus, the pharmacological effect of VD3 was partially mediated by the modulation of DSG3 expression through a simple pathway, without either new transcription or protein synthesis being necessary.