Intracellular redox balance may affect nutrient metabolism in skeletal muscle. Astaxanthin, a carotenoid contained in various natural foods, exerts high antioxidative capacity in the skeletal muscles. The present study investigated the effect of astaxanthin on muscle lipid metabolism in exercise. ICR mice (8 weeks old) were divided into four different groups: sedentary, sedentary treated with astaxanthin, running exercise, and exercise treated with astaxanthin. After 4 weeks of treatment, exercise groups performed treadmill running. Astaxanthin increased fat utilization during exercise compared with mice on a normal diet with prolongation of the running time to exhaustion. Colocalization of fatty acid translocase with carnitine palmitoyltransferase I (CPT I) in skeletal muscle was increased by astaxanthin. We also found that hexanoyl-lysine modification of CPT I was increased by exercise, while astaxanthin prevented this increase. In additional experiment, we found that astaxanthin treatment accelerated the decrease of body fat accumulation with exercise training. Our results suggested that astaxanthin promoted lipid metabolism rather than glucose utilization during exercise via CPT I activation, which led to improvement of endurance and efficient reduction of adipose tissue with training.