We recently discovered that chronic high fructose intakes by lactating rats prevented adaptive increases in rates of active intestinal Ca²⁺ transport and in levels of 1,25-(OH)₂D₃, the active form of vitamin D. Since sufficient Ca²⁺ absorption is essential for skeletal growth, our discovery may explain findings that excessive consumption of sweeteners compromises bone integrity in children. We tested the hypothesis that 1,25-(OH)₂D₃ mediates the inhibitory effect of excessive fructose intake on active Ca²⁺ transport. First, compared with those fed glucose or starch, growing rats fed fructose for 4 wk had a marked reduction in intestinal Ca²⁺ transport rate as well as in expression of intestinal and renal Ca²⁺ transporters that was tightly associated with decreases in circulating levels of 1,25-(OH)₂D₃, bone length, and total bone ash weight but not with serum PTH. Dietary fructose increased the expression of 24-hydroxylase (CYP24A1) and decreased that of 1α-hydroxylase (CYP27B1), suggesting that fructose might enhance the renal catabolism and impair the synthesis, respectively, of 1,25-(OH)₂D₃. Serum FGF23, which is secreted by osteocytes and inhibits CYP27B1 expression, was upregulated, suggesting a potential role of bone in mediating the fructose effects on 1,25-(OH)₂D₃ synthesis. Second, 1,25-(OH)₂D₃ treatment rescued the fructose effect and normalized intestinal and renal Ca²⁺ transporter expression. The mechanism underlying the deleterious effect of excessive fructose intake on intestinal and renal Ca²⁺ transporters is a reduction in serum levels of 1,25-(OH)₂D₃. This finding is significant because of the large amounts of fructose now consumed by Americans increasingly vulnerable to Ca²⁺ and vitamin D deficiency.