Abstract: Objective: This study's goal was to produce walnut peptides by biotransforming walnut meal. It afterwards looked at how well these peptides relieve fatigue and how they influence the gut microbiota of mice that have been fatigued by exercise. Methods: The process for preparing walnut peptide was optimized by a cuniform experimental strategy. A mouse model of exercise weariness was used to evaluate how well walnut peptide relieved fatigue. Additionally, high-throughput sequencing of 16S rRNA was used to examine the impact of walnut peptides on the gut microbiota of exercise-fatigued mice. Results: The optimal process conditions for the preparation of walnut peptides via Yunzhi fermentation were determined to be a 2.5% seed liquor inoculum, a solid-liquid ratio of 1:4, a fermentation duration of 14 days, and a protein content in the walnut meal polypeptide (WPCV) of 87.83±0.55 mg/g. Compared to the model group, mice in both the medium- and high-dose WPCV groups exhibited significantly prolonged times to weight-bearing swimming exhaustion, markedly reduced serum urea nitrogen levels, and notably increased hepatic glycogen content. Additionally, the area under the blood lactate curve was significantly lower in the high-dose WPCV group. Furthermore, WPCV significantly elevated serum lactate dehydrogenase (LDH) and superoxide dismutase (SOD) levels in exercise-induced fatigued mice, although it did not have a significant impact on creatine kinase (CK). On the contrary, WPCV can significantly upregulate the abundance of Akkermansia and Desulfovibrio, while reducing the abundance of Alistipes. Conclusion: WPCV has the effect of relieving fatigue. WPCV can regulate the structure and diversity of gut microbiota in mice, promote the growth of beneficial genera and inhibit the growth of harmful genera, thus accelerating the removal of lactic acid and free radicals in the body, and achieving the effect of relieving physical fatigue. This provides theoretical support for the development of walnut peptides in precision nutritional functional foods.