Abstract: In this study,the anti-fatigue,anti-oxidation and modulating gut microbiota effects of enzymatic hydrolysate of tuna dark meat(EH-TDM)were investigated in mice. The EH-TDM was firstly prepared,the peptides components and abundance were measured via MALDI-TOF/TOF-MS,and the anti-fatigue,anti-oxidation and modulating gut microbiota effects were explored via EH-TDM treatment experiment for 8 weeks in mice. As shown in the results,firstly,most of the peptides contained arginine and branched-chain amino acids(leucine,isoleucine,valine),which showed anti-fatigue effects. Secondly,EH-TDM treatment increased the rolling stick time by 72.8% and 70.8% in male and female mice,respectively. At the same time,EH-TDM treatment in male mice decreased the phosphocreatine(CK)activity and lactic acid(LD)contents in serum by 12.1% and 18.7%,and increased the activity of lactate dehydrogenase(LDH)by 29.8%,all these three indexes showed close relationships with anti-fatigue function. In addition,EH-TDM treatment also decreased the MDA contents in serum by 48.6%,and increased the activity of GSH-Px and SOD by 38.4% and 13.9%,which were involved in anti-oxidation function. On the other hand,EH-TDM treatment in female mice decreased the CK activity and LD content in serum by 3.7% and 12.4%,increased the activity of LDH by 13%,whereas increased the activity of GSH-Px and SOD by 4.99% and 1.74%,and decreased the MDA content by 39.6%. Otherwise,similar change trends of these indexes were observed in liver. Thirdly,EH-TDM treatment decreased the bacterial richness in both male and female mice,whereas only decreased the bacterial diversity in female mice. Moreover,different change profiles were observed in the phylum Bacteroidetes and Firmicutes in mice,and various benefit strains,such as Lactobacillus and short-chain fatty acid-producing bacteria,were increased after EH-TDM treatment,whereas only three key OTUs were detected in both male and female mice.