Currently, there is an urgent need to develop a novel and practical therapeutic approach to accelerate the healing of acute wounds. Mesenchymal stem cell (MSCs)-based therapy is emerging as a promising therapeutic approach for acute skin wounds. However, there are still challenges in clinical application of this strategy, such as low survivability, low retention time and less engraftment in skin wounds.Wharton's jelly mesenchymal stem cells (WJMSCs) were seeded into 3D gelatin microspheres (GM) to identify the biocompatibility of GM. WJMSCs embedded in GM and then encapsulated with Pluronic F-127 (PF-127) and sodium ascorbyl phosphate (SAP) combination to transplant onto acute full-thickness skin wound in mice. Histology, immunohistochemistry and immunofluorescence assay were used to investigate the skin wound healing, dermis regeneration, collagen deposition, cell proliferation and neovascularization.3D GM had strong biocompatibility, comparing with two-dimensional adherent culturing, GM loading increased the cell viability and proliferation ability of WJMSCs. Transplantation of WJMSCs+GM+PF-127+SAP increased the skin wound healing rate, dermis regeneration and type III collagen deposition through improving macrophage polarization, cell proliferation, neovascularization, cell retention and engraftment at skin wound site.The effective three-dimensional encapsulation technology for WJMSCs solved the main problems of cell activity and residence time during MSCs transplantation. WJMSCs+GM+PF-127+SAP transplantation will be a new effective MSCs-biomaterials based therapeutic strategy for acute skin traumatic wounds.WJMSCs+GM+PF-127+SAP transplantation facilitated the acute full-thickness skin wound healing and regeneration, might be a new and effective therapy for acute skin traumatic wounds.