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Fatigue is a common health issue in today's society worldwide. Finding anti-fatigue functional substances seems warranted to alleviate fatigue-related issues. The Lateolabrax japonicuspeptides (LPH) obtained viaenzymatic hydrolysis were identified being rich in Glu, Gly, and hydrophobic amino acids, which exhibited antioxidant properties. However, other biological functions of LPH have yet to be reported. This study main goal was to investigate the anti-fatigue effects of LPH viaa fatigue mice model, and further to elucidate its underlying mechanism using in vivoassay. The results indicated that LPH exhibited strong antioxidant activity and stability in vitro. Meanwhile, LPH significantly extended the swimming time of mice, while also decreasing fatigue marker levels (LA, BUN, CK, and MDA) in serum concurrently by increased levels of antioxidant enzymes, blood glucose, and Na+-K+-ATPase. Histopathological examination revealed that LPH effectively improved tissue damage caused by fatigue. Mechanistically, LPH upregulated the gene and protein expression of Nrf2 and AMPK, indicating its regulatory role in Keap1/Nrf2 and AMPK/SIRT1/PGC-1α/NRF1 signaling pathways. Furthermore, the study demonstrated that LPH could modulate the abundance and balance of fatigue-related gut microbiota (Butyricimonas, Helicobacter, Monoglobus, and Turicibacter). In summary, these findings indicated that LPH exerted its anti-fatigue effect by improving energy metabolism and reducing oxidative stress, suggesting its potential as a promising functional anti-fatigue nutraceutical in food industry.