A recent integrative analysis using a phosphoproteomic approach identified FAM129B, also known as MINERVA, as a downstream effector of the MAP kinase pathway in human melanoma cells. FAM129B protein, which is a member of a small family of proteins, was also found to suppress TNFα/cycloheximide-induced apoptosis in HeLa cells. To investigate the physiological functions of Fam129b in vivo, we generated gene-targeted Fam129b-mutant mice in which, the amino terminal coding exon was replaced by lacZ. We found that homozygous mutant mice are viable and fertile and that Fam129b is considerably expressed in most of the epidermal keratinocytes of both embryonic and adult mice. Although no gross defect was observed in the skin of the Fam129b-deficient mice, wound healing subsequent to skin puncturing was significantly delayed. Furthermore, overexpression of Fam129b promoted HaCaT cell motility in an N-terminal pleckstrin homology domain-dependent manner, but not proliferation. Microarray analysis of the Fam129b transfectant exhibited substantial upregulation of several genes related to wound repair and cell motility. These results suggest that expression of Fam129b in epidermal keratinocytes accompanied by alteration of wound healing-related gene expression is necessary for regulation of cell motility and thereby, contributes to the appropriate wound healing process.