Abstract <p>Wnt/β-catenin signaling pathway is an important cell-cell communication system, which plays an indispensable role in skin wound repair by stimulating cell proliferation, dedifferentiation and tissue remodeling. Meanwhile, R-spondin1 (RSPO1), as a classic Wnt agonist, can significantly enhance the activity of the Wnt/β-catenin signaling pathway. Human umbilical cord mesenchymal stem cells (UCMSC) are regulatory cells that accelerate wound healing primarily through the release of paracrine signaling molecules, but the underlying effects of RSPO1-activated UCMSC-conditioned medium (UCMSC-CM) on skin wound repair have not been clearly defined. In our study, we investigated the role of RSPO1-activated UCMSC-conditioned medium on the biological activities of human umbilical vein endothelial cells (HUVECs), which are widely used as a model for studying angiogenesis related to wound healing. After determining the optimal action condition of RSPO1 on UCMSC and the protein concentration of RSPO1-activated UCMSC-CM on HUVECs by the Cell Counting Kit-8 (CCK-8) method, we comprehensively evaluated the effect of RSPO1-activated UCMSC-CM on the biological functions of HUVECs by BrdU proliferation assay, tubule formation assay and scratch assay, in order to provide new therapeutic ideas for skin wound healing. Our findings revealed that, in comparison to the non-RSPO1-activated control group, RSPO1-activated UCMSC-CM exhibited more robust stimulatory effects on HUVEC proliferation, angiogenesis, and cell migration. This suggests that targeting RSPO1-activated UCMSC-CM may represent a promising strategy for enhancing wound healing outcomes.</p>