S100 calcium-binding protein A8 (S100A8) and S100A9 are small molecular weight calcium-binding regulatory proteins that have been involved in multiple chronic inflammatory diseases. However, the role of S100A8 and S100A9 in keratinocytes in wounded skin and how they are regulated during this process are still unclear. Here, we found that S100A8 and S100A9 were both upregulated in burn-wounded skins in vivo and thermal-stimulated epidermal keratinocytes in vitro, accompanied by increased levels of epithelial-mesenchymal transition (EMT). Then, we demonstrated that upregulation of S100A8 and S100A9 alone or together enhanced characteristics of EMT in normal keratinocytes, manifested by excessive proliferation rate, abnormal ability of cell invasion, and high expression levels of EMT marker proteins. The transcription factor PU box-binding protein (PU.1) bound to the promoter regions and transcriptionally promoted the expression of S100A8 and S100A9 both in the human and mice, and it had strong positive correlations with both S100A8 and S100A9 protein levels in burned skin in vivo. Moreover, PU.1 positively regulated expression of S100A8 and S100A9 in a dose-dependent manner, and enhanced EMT of keratinocytes in vitro. Finally, through the burn mouse model, we found that PU.1-/- mice displayed a lower ability of scar formation, manifested by smaller scar volume, thickness, and collagen content, which could be enhanced by S100A8 and S100A9. In conclusion, PU.1 transcriptionally promotes expression of S100A8 and S100A9, thus positively regulating epithelial-mesenchymal transformation (EMT) and invasive growth of dermal keratinocytes during scar formation post burn.