Objective: This study aims to investigate the structural and functional characteristics of walnut protein hydrolysates (WPHs) with different molecular weights prepared using protease from Dregea sinensis Hemsl, as well as the anti-fatigue effects of low-molecular-weight walnut protein hydrolysates (LWPs) and their impact on the cecal microbiota and faecal metabolism of mice. Methods: The anti-fatigue activity of WPHs with different molecular weights was evaluated, and the LWPs were analyzed in a centralized manner. A 28-day gavage study was conducted to assess LWP’s anti-fatigue benefits in mice, supplemented by metabolomic analysis to explore its impact on metabolic pathways. Results: Our findings revealed that LWP significantly outperformed unhydrolyzed walnut protein (WP) in terms of water retention, lipid retention, emulsifying properties, and foaming capacity. Notably, differential protein expression associated with LWP highlighted pathways related to antioxidant activity. In vivo studies showed that LWP markedly enhanced glycogen storage in the muscles and liver of mice, while reducing serum levels of serum urea nitrogen, lactate dehydrogenase, blood lactic acid, and creatine kinase. Furthermore, the levels of Superoxide Dismutase and Glutathione were significantly elevated, alongside a reduction in Malondialdehyde, indicating that LWP’s anti-fatigue effect is closely linked to improved oxidative stress resistance. Additionally, LWP promoted beneficial increases in microbial populations such as Akkermansia, Alistipes, Eubacterium, and Muribaculum, which are associated with enhanced fatigue resistance. Metabolomic analysis indicated significant enrichment in glycerophospholipid metabolism and amino acid biosynthesis, identifying key metabolites including palmitoylethanolamide and 4-methyl-5-thiazoleethanol, both of which are integral to health maintenance. Conclusions: LWP demonstrates a robust anti-fatigue effect, supported by its accessibility, straightforward preparation, and eco-friendly characteristics. These attributes suggest that LWP has promising potential for inclusion in health products aimed at enhancing vitality and combating fatigue.