A magnesium (Mg)-deficient diet results in decreased serum phosphorus (P) levels and increased urinary P excretion; however, the mechanisms responsible for these effects are unclear. Fibroblast growth factor-23 (FGF-23) is a potent regulator of P homeostasis. To determine the mechanisms responsible for the change in serum levels and urinary excretion of P with Mg deficiency, the present study examined the effects of Mg deficiency on serum FGF-23 levels. Male rats were randomized by weight into two groups and fed a control diet (Mg concentration: 0.05%) or a Mg-deficient diet (Mg concentration: Mg-free) for 21 days. Serum P levels in rats fed the Mg-deficient diet were significantly lower than in rats fed the control diet. Furthermore, urinary P excretion was significantly higher in rats fed the Mg-deficient diet compared to rats fed the control diet. Conversely, the tubular reabsorption rate of P was significantly lower in rats fed the Mg-deficient diet than in the controls. Serum FGF-23 levels in rats fed the Mg-deficient diet were significantly higher than those in animals fed the control diet. The results from the present study indicate that 1) Mg deficiency increases serum FGF-23 levels; and 2) Mg deficiency causes increased urinary P excretion via inhibition of renal P reabsorption, resulting in a lowering of serum P levels. Moreover, we suggest that the high serum FGF-23 levels induced by Mg deficiency contribute to the decrease in renal P reabsorption.