This study investigated the mechanism by which Rheum palmatum polysaccharide (RPS) regulates multiple molecular pathways to enhance skin wound repair. Utilizing in vitro HaCaT cell proliferation and migration assays combined with a murine skin injury model, the effects of RPS on wound healing were evaluated through cell viability tests, scratch assays, histopathological staining, and molecular biology techniques. Results demonstrated that RPS at appropriate concentrations significantly enhanced epidermal cell proliferation and accelerated migration. Concurrently, it upregulated the gene expression of key factors, including tumor necrosis factor-α (TNF-α), epidermal growth factor (EGF), and transforming growth factor-β1 (TGF-β1). Further analysis revealed that RPS activated the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)-mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) (PI3K/AKT-MAPK/ERK) signaling pathway, markedly increasing the phosphorylation levels of key pathway proteins (AKT Ser 473 and ERK Thr 202/Tyr 204) and promoting mRNA expression of collagen type I alpha 1 chain (Col1A1) and collagen type III alpha 1 chain (Col3A1). Animal experiments confirmed that the RPS-treated group exhibited superior epidermal regeneration rates and collagen deposition compared to the control group. Wound tissues in the RPS group showed enhanced expression of growth factors (TNF-α, EGF, TGF-β1) and collagen-related genes (Col1A1, Col3A1), alongside significantly elevated phosphorylation levels of the PI3K/AKT-MAPK/ERK pathway. This study elucidates that RPS synergistically promotes wound repair through a multi-target regulatory mechanism, involving activation of critical signaling pathways, upregulation of repair-related factors, and enhancement of collagen remodeling. These findings provide a vital theoretical foundation for the development of novel wound repair therapeutics.