Fibroblast growth factor 21 (FGF21) might increase glucose uptake in adipocytes and reduce plasma glucose and triglycerides in animal models of diabetes. The aim of the present study was to assess serum FGF21 concentration in lean and obese subjects, its regulation by hyperinsulinemia and relationship with insulin sensitivity and glucose and lipid oxidation. We examined 76 subjects with normal glucose tolerance, 36 lean (BMI <25 kg/m2) and 39 overweight or obese (BMI between 25 and 40 kg/m2). Euglycemic hyperinsulinemic clamp and indirect calorimetry in the baseline state and during last 30 min of the clamp were performed. An increase in respiratory quotient (ΔRQ) in response to insulin was used as a measure of metabolic flexibility. Serum FGF21 was measured in the baseline state and after the clamp. Fasting serum FGF21 did not differ between the lean and obese groups. Hyperinsulinemia resulted in an increase in serum FGF21 in the obese (P=0.0002), but not in the lean group (P=0.33). In consequence, post-clamp serum FGF21 was higher in the obese subjects (P=0.0002). Fasting FGF21 was related to waist circumference (r=0.26, P=0.023), fasting insulin (r=0.31, P=0.01) and triglycerides (r=0.39, P=0.001). Post-clamp FGF21 was associated with BMI (r=0.42), waist circumference (r=0.46, both P<0.001), fasting insulin (r=0.28, P=0.031), lipid oxidation during the clamp (r=0.25, P=0.039), the change in glucose oxidation in response to insulin (r=−0.25, P=0.043) and ΔRQ (r=−0.26, P=0.029). In the obese group, correlations between fasting FGF21 and a change in glucose oxidation (r=−0.35, P=0.034) and lipid oxidation (r=0.32, P=0.049) in response to insulin were observed. Our data show that serum FGF21 is associated with insulin resistance-related abnormalities of human obesity.