Our previous report indicated that magnesium (Mg) deficiency increased molybdenum (Mo) concentration in the rat liver, suggesting the possibility that Mg deficiency affects Mo metabolism. Growing male rats were given a control diet or a Mg-deficient diet for 4 weeks. Urine and feces were collected during the second and fourth weeks of the feeding trial. The liver, kidney, spleen, skeletal muscle, and blood were collected at the end of the feeding trial. Mg deficiency did not affect the apparent absorption of Mo, but it reduced urinary excretion of Mo. The retention of Mo tended to be higher in the Mg-deficient group than in the control group. Hepatic Mo concentration was higher in the Mg-deficient group than in the control group, but Mg deficiency did not affect Mo concentration in other tissues and plasma. Mg deficiency downregulated the mRNA expression of Mo transporter 2 (MOT2) in the liver, but not in the kidney. These results suggest that Mg deficiency decreases urinary Mo excretion, which is too slight to affect plasma Mo concentration, and that Mg deficiency selectively disturbs the homeostatic mechanism of Mo in the liver, which is not related to the mRNA expression of MOT2 in the liver.