AbstractAfter a skin injury, keratinocytes switch from a state of homeostasis to one of regeneration leading to the reconstruction of the epidermal barrier. The regulatory mechanism of gene expression underpinning this key switch during human skin wound healing is enigmatic. Long noncoding RNAs (lncRNAs) constitute a new horizon in the understanding of the regulatory programs encoded in the mammalian genome. By comparing the transcriptome of an acute human wound and skin from the same donor as well as keratinocytes isolated from these paired tissue samples, we generated a list of lncRNAs showing changed expression in keratinocytes during wound repair. Our study focused onHOXC13-AS, a recently evolved human lncRNA specifically expressed in epidermal keratinocytes, and we found that its expression was temporally downregulated during wound healing. In line with its enrichment in suprabasal keratinocytes,HOXC13-ASwas found to be increasingly expressed during keratinocyte differentiation, but its expression was reduced by EGFR signaling. AfterHOXC13-ASknockdown or overexpression in human primary keratinocytes undergoing differentiation induced by cell suspension or calcium treatment and in organotypic epidermis, we found thatHOXC13-ASpromoted keratinocyte differentiation. Moreover, RNA pull-down assays followed by mass spectrometry and RNA immunoprecipitation analysis revealed that mechanisticallyHOXC13-ASsequestered the coat complex subunit alpha (COPA) protein and interfered with Golgi-to-endoplasmic reticulum (ER) molecular transport, resulting in ER stress and enhanced keratinocyte differentiation. In summary, we identifiedHOXC13-ASas a crucial regulator of human epidermal differentiation.