Objective To investigate the active mechanism of Ejiao powder, prepared from donkey-hide gelatin, rock sugar, and rice wine, in alleviating physical fatigue in mice.Methods Fifty specific pathogen-free (SPF) male BALB/c mice were randomly divided into five groups (10 in each group): a blank negative control group, a model positive control group, and three experimental groups receiving low (0.67 g/kg), medium (1.33 g/kg), and high (2.67 g/kg) doses of Ejiao powder. Except for the control group, all other groups were administered different doses of Ejiao powder via gavage for 30 consecutive days. Following administration, weight-bearing swimming time was recorded, and post-exercise serum urea, muscle glycogen, blood lactic acid, and ATP levels were measured. Additionally, the microbial community in fecal samples was analyzed through genome sequencing.Results Compared with the model control group, the low-dose Ejiao powder group exhibited a significant increase in weight-bearing swimming time (P<0.05). Serum urea levels after exercise were significantly reduced in all Ejiao-treated groups (P<0.01), while muscle glycogen reserves in the medium-and high-dose groups showed significant differences (P<0.01). Blood lactic acid levels were significantly decreased (P<0.01), and ATP metabolism was significantly enhanced in the medium-and high-dose groups (P<0.01). Genome sequencing of fecal microbiota revealed a ranking of microbial diversity as follows: medium-dose group>low-dose group>high-dose group>blank control group. Mice in the low-and high-dose Ejiao powder groups exhibited a reduction in Bacteroidetes, while those in the medium-dose group showed a significant increase in Firmicutes, which corresponded to improved carbohydrate and lipid metabolism, providing more energy to alleviate physical fatigue.Conclusion Ejiao powder demonstrated an active effect in alleviating physical fatigue in mice and improved microecological imbalance.