The purpose of the present study was to determine whether fructose is the nutrient mediator of sucrose-induced insulin resistance and glucose intolerance. Toward this end, male rats were fed a purified starch diet (68% of total calories) for a 2-wk baseline period. After this, rats either remained on the starch (ST) diet or were switched to a sucrose (SU, 68% of total calories), fructose/glucose (F/G, 34/34% of total calories), or fructose/starch (F/ST, 34/34% of total calories) diet for 5 wk. Rats then underwent either an intravenous glucose tolerance test (n = 10/diet) or a euglycemic, hyperinsulinemic clamp (n = 8 or 9/diet). Incremental glucose and insulin areas under the curve in SU, F/G, and F/ST were on average 61 and 29% greater than ST, respectively, but not significantly different from one another. During clamps, glucose infusion rates (mg · kg⁻¹ · min⁻¹) required to maintain euglycemia were significantly lower (P< 0.05) in SU, F/G, and F/ST (13.4 ± 0.9, 9.5 ± 1.7, 11.3 ± 1.3, respectively) compared with ST (22.8 ± 1.1). Insulin suppression of glucose appearance (mg · kg⁻¹ · min⁻¹) was significantly lower (P < 0.05) in SU, F/G, and F/ST (5.6 ± 0.5, 2.2 ± 1.2, and 6.6 ± 0.7, respectively) compared with ST (9.6 ± 0.4). Insulin-stimulated glucose disappearance (mg · kg⁻¹ · min⁻¹) was significantly lower (P < 0.05) in SU, F/G, and F/ST (17.9 ± 0.6, 16.2 ± 1.3, 15.3 ± 1.8, respectively) compared with ST (24.7 ± 1.2). These data suggest that fructose is the primary nutrient mediator of sucrose-induced insulin resistance and glucose intolerance.