Retinol-binding protein 4 (RBP4) is a plasma protein which is elevated in obesity and type 2 diabetes. We aimed to investigate whether RBP4 represents a mechanism underlying the associations between low birth weight (LBW), high-fat diet, and insulin resistance. Forty-six young, lean men with low (n = 20) or normal (n = 26) birth weight underwent a 5-day high-fat high-calorie (HFHC) dietary intervention. In vivo glucose metabolism was assessed by euglycemic-hyperinsulinemic clamp, glucose tracer and intravenous glucose tolerance test techniques. Body composition was measured by a dual-energy x-ray absorptiometry scan, and plasma RBP4 by an enzyme-linked immunosorbent assay. RBP4 was not associated with birth weight, but with BMI (β = 0.9 µg/ml (0.08; 1.8) (95% confidence interval), P = 0.03) and plasma levels of low-density lipoprotein cholesterol (β = 5.3 µg/ml (1.9; 8.7), P = 0.03) and triglycerides (β = 15.4 µg/ml (9.5; 21.3), P < 0.001). Under baseline diet conditions, RBP4 was associated with decreased disposition index (D(i)) (β = -2.4% (-4.5%; -0.2%), P = 0.04) and increased basal hepatic glucose production rate (HGP) (β = 0.02 mg kg(-1) min(-1) (0.002; 0.04), P = 0.03), but not associated with peripheral glucose disposal rate or hepatic insulin resistance index. RBP4 levels were not influenced by overfeeding or related to peripheral and hepatic insulin resistance provoked by the dietary intervention. In conclusion, plasma RBP4 in young men associates with components of the metabolic syndrome, but is not determined by birth weight and seems not to be involved in short-term high-fat diet-induced insulin resistance.