We investigated the effect of exercise on iron metabolism in horses. Four horses were walked on a mechanical walker for 1 wk (pre-exercise). They then performed moderate exercise on a high-speed treadmill in the first week of the exercise and relative high in the second week and high in the third week. Serum iron was significantly lower in the third week of exercise than in the pre-exercise. Transferrin saturation (TS) was significantly lower in the first and third weeks of exercise than in the pre-exercise. Serum haptoglobin was significantly lower in the first week of exercise than in the pre-exercise and further significantly lower in the second and third weeks than in the first. The packed cell volume did not change during the experiment. The exercise significantly increased the apparent absorption of iron. Urinary iron excretion did not change throughout the experiment. Sweat iron loss did not change during the exercise. The exercise significantly increased iron balance. We considered that hemolysis is induced by moderate exercise and is further enhanced by heavy exercise, which decreases serum iron and TS. However, the increase in iron absorption compensates for the adverse effect of exercise on iron status. Therefore, exercise does not induce anemia in horses.