Skeletal muscle injuries are prevalent in competitive sports, often disrupting elite athletes' training programs. Platelet-rich plasma (PRP) is abundant in growth factors, cytokines, and chemokines, making it a potential solution for treating muscle injuries. Adipose-derived stem cells (ADSCs) therapy presents promise for tissue regeneration, given their self-renew and differentiation capabilities. This study aims to investigate the impact of PRP on ADSCs, with a focus on cell migration. ADSCs were isolated from the subcutaneous adipose tissue of Sprague Dawley rats. Subsequently, these ADSCs were treated with PRP. The migration ability was assessed through in vitro wound assays, migration assays, and cell spreading assays. Molecular analysis, employing Western blot, was performed to examine the expression levels of phosphorylated focal adhesion kinase (FAK), FAK, phosphorylated paxillin, paxillin, and vinculin. The filamentous actin (F-actin) cytoskeleton formation was assessed through immunofluorescence staining. The study revealed a positive impact of PRP on ADSC migration in the in vitro wound assay, migration assay, and cell spreading assay. PRP elevated the expression levels of phosphorylated FAK, FAK, phosphorylated paxillin, paxillin, and vinculin in ADSCs. Additionally, PRP affected the distribution of F-actin in ADSCs. Furthermore, siRNA-mediated knockdown of FAK and paxillin confirmed their essential roles in PRP-enhanced ADSC migration. In conclusion, our findings demonstrated that PRP promoted the migration, spreading, in vitro wound healing, and cytoskeleton rearrangement of ADSCs by upregulating the expressions of phosphorylated FAK, FAK, phosphorylated paxillin, paxillin, and vinculin. This insight supports the potential use of PRP in combination with ADSCs for regeneration therapies.