Objectives: This study aimed to clarify the effect of lactic acid bacteria-fermented corn protein hydrolysate (FCH) on fatigue in mice and explore the connection between fatigue-related indicators and intestinal microbial flora. Methods: The fatigue model of mice was constructed by exercise endurance experiment. The anti-fatigue level of FCH was evaluated by measuring physiological and biochemical indexes in mouse serum, liver and skeletal muscle. The relationship between FCH, intestinal flora and fatigue was explored through the analysis of intestinal microbial diversity in mice, and the anti-fatigue mechanism of FCH was further analyzed. Results: The results showed that the weight-bearing swimming time of mice was prolonged by 1.96 times, and the running time of mice was prolonged by 2.63 times in the high-dose FCH (FCH-H) group. Moreover, the lactic acid contents in the blood were reduced by 16.00%, and lactate dehydrogenase activity and urea nitrogen contents basically returned to the normal level. Meanwhile, the malondialdehyde contents were reduced by 31.24%, and superoxide dismutase activity and glutathione contents were increased by 1.84 times and 1.72 times, respectively. In addition, the glycogen contents of the body were restored, and the muscle glycogen and liver glycogen were increased by 1.81 and 5.81 times, respectively. Analysis of intestinal microbial flora diversity in mice showed that the highest relative abundance was Lactobacillus, and the FCH group could recover and even increase its relative abundance. Lactobacillus was significantly positively correlated with muscle glycogen and SOD. Conclusions: FCH can alleviate fatigue by regulating fatigue-related indicators and improving the intestinal microbial flora of the organism.