As a precious traditional Chinese medicine, Asini Corii Colla (ACO) has long been used for hematopoiesis, hemostasis, and wound repair, yet its mechanisms in wound healing remain incompletely elucidated. Using human skin fibroblasts (HSF) and a murine macrophage-like cell line (RAW 264.7), along with a rat full-thickness skin defect model, the biocompatibility, cell proliferation and migration-promoting ability, and wound healing acceleration efficacy of ACO were investigated. HSF cells were used to assess proliferation and migration, whereas RAW 264.7 cells were used for cytocompatibility and macrophage polarization studies. The potential mechanisms were further explored by transcriptomic analysis. In vitro cytotoxicity assays revealed that ACO exhibited no obvious cytotoxicity at concentrations up to 100 mg/mL. Accordingly, further functional assays demonstrated that within the safe concentration range of 10-40 mg/mL, ACO significantly promoted acute wound healing. H&E and Masson’s trichrome histological staining, IL-1β and IL-10 immunohistochemistry, and CD31/α-SMA immunofluorescence staining revealed that ACO enhanced epithelial regeneration, increased collagen deposition, improved collagen fiber arrangement, effectively downregulated the pro-inflammatory cytokine IL-1β, upregulated the anti-inflammatory cytokine IL-10, and significantly increased CD31-positive vessel density. Transcriptomic analysis further revealed temporally distinct associations of ACO: at early stages, ACO treatment was associated with gene signatures related to cell migration, inflammatory regulation, and matrix synthesis, whereas at later stages it was associated with signatures related to tissue contraction, vascular maturation, and metabolic reprogramming. These findings provide preliminary evidence that ACO may be a promising bioactive ingredient for functional skin repair, warranting exploration of its synergistic potential for advanced wound management.