Objectives: this study aimed to explore the effects of different intake levels (20–80%) of highland barley on the anti-fatigue capacity of ICR mice, focusing on energy metabolism, metabolite accumulation, oxidative stress, and changes in the gut microbiota. Methods: male ICR mice were assigned to five groups: control (normal diet) and four experimental groups with highland barley supplementation at 20%, 40%, 60%, and 80% of total dietary energy. Anti-fatigue performance was assessed by behavioral experiments (rotarod, running, and exhaustive swimming tests), biochemical markers, and gut microbiota analysis. Results: the results showed that moderate supplementation (20%) significantly enhanced exercise endurance and anti-fatigue capacity, as evidenced by increased liver glycogen (134.48%), muscle glycogen (87.75%), ATP content (92.07%), Na⁺-K⁺-ATPase activity (48.39%), and antioxidant enzyme activities (superoxide dismutase (103.31%), catalase (87.75%), glutathione peroxidase (81.14%). Post-exercise accumulation of blood lactate, quadriceps muscle lactate, serum urea nitrogen, and the oxidative stress marker malondialdehyde was significantly reduced, with differences of 31.52%, 21.83%, 21.72%, and 33.76%, respectively. Additionally, 20% supplementation promoted the growth of beneficial gut microbiota associated with anti-fatigue effects, including unclassified_f_Lachnospiraceae, g_norank_f_Peptococcaceae, Lachnospiraceae NK4A136, Colidextribacter, and Turicibacter. However, when intake reached 60% or more, anti-fatigue effects diminished, with decreased antioxidant enzyme activity, increased accumulation of metabolic waste, and a rise in potentially harmful microbiota (Allobaculum, Desulfovibrio, and norank_f_norank_o_RF39). Conclusions: moderate highland barley supplementation (20% of total dietary energy) enhances anti-fatigue capacity, while excessive intake (≥60%) may have adverse effects.