Recently, it has been reported that mesenchymal stem cell (MSC)-derived humoral factors promote skin wound healing. As these humoral factors are transiently stored in cytoplasm, we collected them as part of the cell extracts from MSCs (MSC-ext). This study aimed to investigate the effects of MSC-ext on skin wound healing. We examined the effects of MSC-ext on cell proliferation and migration. Additionally, the effect of MSC-ext on skin wound healing was evaluated using a mouse skin defect model. The MSC-ext enhanced the proliferation of dermal fibroblasts, epithelial cells, and endothelial cells. It also increased the number of migrating fibroblasts and epithelial cells. The skin defects treated with MSC-ext demonstrated rapid wound closure compared to those treated with phosphate-buffered saline. The MSC-ext group exhibited a thicker dermis, larger Picrosirius red-positive areas, and a higher number of Ki67-positive cells. Our results indicate that MSC-ext promotes the proliferation and/or migration of fibroblasts, epithelial cells, and endothelial cells, and enhances skin wound healing. This suggests the therapeutic potential of MSC-ext in treating skin defects as a novel cell-free treatment modality.