Abstract: Objective: Based on network pharmacology combined with the GEO database and in vivo and in vitro experiments to explore the potential mechanisms of litchi semen extract (LZH) for preventing exercise-induced muscle damage (EIMD). Methods: Hematoxylin-eosin (HE) staining and comparison of the levels of indicators of skeletal muscle damage in serum were performed to detect the effectiveness of LZH in preventing EIMD. The main active ingredients of LZH were searched through the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database in conjunction with the published literature, and the targets corresponding to the active ingredients were found through the relevant websites, which were intersected with the EIMD-related targets obtained from the GEO database. The STRING database screened the intersected targets and finally enriched them for analysis. The results of the network pharmacology predictions were validated in vivo using the centrifugal exercise-induced EIMD mouse model and in vitro using the H₂O₂-induced C2C12 cell model. Results: In vivo experiments showed that skeletal muscle fiber cross-sectional area (P<0.05), creatine kinase (CK) (P<0.001) and lactate dehydrogenase (LDH) (P<0.01) were significantly reduced after the intervention of LZH. The results of network pharmacology showed that LZH had 14 active ingredients, corresponding to 367 targets. The GEO database obtained 1015 targets related to EIMD. The intersection of the two yielded 37 intersecting targets. The enrichment analysis demonstrated p53-mediated cell cycle arrest as the primary target for validation. In vivo experiments showed that LZH significantly reduced the expression of p53 (P<0.01), p21 (P<0.001), BCL2-associated X (Bax) (P<0.05), and the expression of Cyclin D1 (P<0.05), B-cell lymphoma-2 (Bcl-2) (P<0.05) expression was significantly increased. In vitro experiments showed that low and high dose pretreatment of LZH significantly decreased the expression of apoptosis (P<0.001), p53 (P<0.05), p21 (P<0.01, P<0.001) and increased the expression of Cyclin D1 (P<0.05, P<0.001). Conclusion: LZH attenuates EIMD, and its mechanism is closely related to the activation of G1 phase arrest and attenuation of apoptosis in skeletal muscle cells. The results suggest that LZH can be used as a nutritional supplement to prevent EIMD.