Glucokinase (GK) is required for cellular glucose sensing, although there is a paucity of data regarding its role in the counterregulatory response to hypoglycemia in humans. Because fructose has been shown to modulate GK activity, we examined the effects of an acute infusion of fructose on hypoglycemia counterregulation in seven lean nondiabetic subjects. Using stepped hypoglycemia clamp studies (5.0, 4.4, 3.9, and 3.3 mmol/l target plasma glucose steps, 50 min each), subjects were studied on two separate occasions, without (control) or with co-infusion of fructose (1.2 mg · kg⁻¹ · min⁻¹). Fructose induced a resetting of the glycemic thresholds for secretion of epinephrine (3.8 ± 0.1 mmol/l) and glucagon (3.9 ± 0.2 mmol/l) to higher plasma glucose concentrations (4.0 ± 0.1 mmol/l [P = 0.006] and 4.1 ± 0.1 mmol/l [P = 0.03], respectively). In addition, the magnitude of increase in epinephrine and glucagon concentrations was higher after administration of fructose (48 and 39%, respectively, P < 0.05 for both). The amplification of these hormonal responses was specific because plasma norepinephrine, growth hormone, and cortisol were comparable in both sets of studies. Endogenous glucose production, measured with [3-³H]glucose, increased by 47% (P < 0.05) in the fructose infusion studies compared with 14% (P = NS) in the control studies. In addition, glucose uptake was more suppressed with fructose infusion (by 33%, P < 0.05). In concert with these effects of fructose on glucose kinetics, average glucose infusion rate was markedly reduced in the fructose infusion studies during the 3.9-mmol/l glucose step (4.6 ± 0.9 vs. 7.4 ± 1.1 μmol · kg⁻¹ · min⁻¹, respectively, P = 0.03) and during the 3.3-mmol/l glucose step (0.5 ± 0.1 vs. 5.2 ± 1.2 μmol · kg⁻¹ · min⁻¹, respectively, P < 0.001), suggesting more potent glucose counterregulation and improved recovery from hypoglycemia with fructose infusion. We conclude that infusion of a catalytic dose of fructose amplifies the counterregulatory response to hypoglycemia by both increases in hormonal activation and augmentation of glucose counterregulation in humans.