Regulation of human keratinocyte (HK) migration is critical for skin wound healing. Profiling HK migration-specific genes could help us gain a comprehensive understanding of the process. The main challenge is to separate genes that are unrelated to migration, but simultaneously induced by the same growth factor. In this study, we took advantage of a unique response of HKs to transforming growth factor-beta (TGF-beta), which inhibits proliferation but not migration of HKs, to suppress selectively the proliferation-related genes. Furthermore we stimulated HKs independently with TGF-alpha or insulin and identified the common genes and eliminated TGF-alpha- or insulin-specific genes. Under these conditions, we obtained profiles of the immediate-early genes (IEGs, at 30 minutes), early genes (EGs, at 60 minutes), and delayed-early genes (DEGs, at 120 minutes) by microarray analyses, followed by quantitative real-time reverse transcription-PCR (QRT-PCR) validation and functional characterization by RNA interference (RNAi). Our results revealed the following: (1) 25 upregulated and 1 downregulated IEGs; (2) 58 upregulated and 15 downregulated EGs, and (3) 13 upregulated and 3 downregulated DEGs in both TGF-alpha- and insulin-stimulated HKs. Three genes, all encoding secreted molecules, were investigated in HK migration. These cell motility-specific gene profiles may prove useful to skin wound healing.