The present study was designed to investigate whether L-arginine (Arg) supplementation in exercise affects nitric oxide (NO) synthesis in tissues and thus iron metabolism. Rats were assigned to one of four groups: EG (Exercise), SG (Sedentary), EAG (Exercise + Arg), and SAG (Sedentary + Arg). Both EG and EAG swam 2 h/day for 3 months. Both SAG and EAG received 3% Arg supplementation in their drinking water. The results showed that Arg supplementation in exercise (EAG) significantly increased nitrite and nitrate (NOx) concentration in the kidney and BMC, rather than in the liver, spleen and heart. Arg supplementation significantly increased both nonheme iron (NHI) and catalytic iron (CI) content in the kidney, to the extent that the ratio of CI/NHI or storage iron (SI)/NHI was not significantly affected, and significantly decreased NHI content and increased CI content in BMC, to the extent that SI content or SI/NHI was significantly decreased. These findings suggest that Arg supplementation in exercise, possibly through increasing NO synthesis, may change CI formation in the kidney and BMC, and affect iron storage in BMC rather than in the kidney.