Fructose consumption by Americans has increased markedly whereas Ca²⁺ intake has decreased below recommended levels. Since fructose metabolism decreases enterocyte ATP concentrations, we tested the hypothesis that luminal fructose acutely reduces active, diet-inducible Ca²⁺ transport in the small intestine. We confirmed that the decrease in ATP concentrations was indeed greater in fructose- compared to glucose-incubated mucosal homogenates from wildtype, and was prevented in fructose-incubated homogenates from ketohexokinase (KHK)-/-, mice. We then induced active Ca²⁺ transport by chronically feeding wildtype, fructose transporter GLUT5-/-, as well as KHK-/- mice, a low Ca²⁺ diet, and measured transepithelial Ca²⁺ transport in everted duodenal sacs incubated in solutions containing glucose, fructose or their nonmetabolizable analogs. The diet-induced increase in active Ca²⁺ transport was proportional to dramatic increases in expression of the Ca²⁺-selective channel TRPV6 as well as of the Ca2+ binding protein CaBP9k, but not that of the voltage-dependent L-type channel Ca(v)1.3. Crypt-villus distribution of CaBP9k seems heterogeneous, but low Ca²⁺ diets induce expression in more cells. In contrast, KHK distribution is homogeneous, suggesting that fructose metabolism can occur in all enterocytes. Diet-induced Ca2+ transport was not enhanced by addition of the enterocyte fuel glutamine, and was always greater in sacs of wildtype, GLUT5-/- and KHK-/- mice incubated with fructose or nonmetabolizable sugars than those incubated with glucose. Thus, duodenal Ca²⁺ transport is not affected by fructose and enterocyte ATP concentrations but instead may decrease with glucose metabolism, as Ca²⁺ transport remains high with 3-O-methylglucose that is also transported by the glucose transporter SGLT1, but cannot be metabolized.