The ATP2A2 gene encodes Ca2+-dependent ATPase, the dysfunction of which causes Darier disease. In this study, we analyzed the promoter structure of the human ATP2A2 gene using primary normal human keratinocytes (NHK). Reporter assays showed that deletion of -550/-529, -488/-472, -390/-362, or -42/-21 resulted in a significant decrease in human ATP2A2 promoter activity. Electrophoretic mobility shift assay (EMSA) showed that Sp1 is a transcription factor that binds to the -550/-529 and -488/-472 regions of the promoter. Chromatin immunoprecipitation (ChIP) assay demonstrated that Sp1, but not Sp3, binds to the promoter region of the ATP2A2 gene in NHK cells in vivo. Knockdown of Sp1 expression by small interfering RNA resulted in a marked reduction in ATP2A2 promoter activity and ATP2A2 mRNA levels in NHK, suggesting that Sp1 positively transactivates the ATP2A2 promoter in NHK. This is early evidence demonstrating that Sp1 plays an important and positive role in ATP2A2 gene expression in NHK in vivo and in vitro.