Elevated blood fructose concentrations constitute the basis for organ dysfunction in fructose-induced metabolic syndrome. We hypothesized that diet-induced changes in blood fructose concentrations are regulated by ketohexokinase (KHK) and the fructose transporter GLUT5. Portal and systemic fructose concentrations determined by HPLC in wild-type mice fed for 7 days 0% free fructose were <0.07 mM, were independent of time after feeding, were similar to those of GLUT5^−/−, and did not lead to hyperglycemia. Postprandial fructose levels, however, increased markedly in those fed isocaloric 20% fructose, causing significant hyperglycemia. Deletion of KHK prevented fructose-induced hyperglycemia, but caused dramatic hyperfructosemia (>1 mM) with reversed portal to systemic gradients. Systemic fructose in wild-type and KHK^−/− mice changed by 0.34 and 1.8 mM, respectively, for every millimolar increase in portal fructose concentration. Systemic glucose varied strongly with systemic, but not portal, fructose levels in wild-type, and was independent of systemic and portal fructose in KHK^−/−, mice. With ad libitum feeding for 12 wk, fructose-induced hyperglycemia in wild-type, but not hyperfructosemia in KHK^−/− mice, increased HbA_1c concentrations. Increasing dietary fructose to 40% intensified the hyperfructosemia of KHK^−/− and the fructose-induced hyperglycemia of wild-type mice. Fructose perfusion or feeding in rats also caused duration- and dose-dependent hyperfructosemia and hyperglycemia. Significant levels of blood fructose are maintained independent of dietary fructose, KHK, and GLUT5, probably by endogenous synthesis of fructose. KHK prevents hyperfructosemia and fructose-induced hyperglycemia that would markedly increase HbA_1c levels. These findings explain the hyperfructosemia of human hereditary fructosuria as well as the hyperglycemia of fructose-induced metabolic syndrome.