Maintaining hyperinsulinemia (∼160 mU/l) during steady-state hypercarnitinemia (∼550 μmol/l) increases skeletal muscle total carnitine (TC) content by ∼15% within 5 h. The aim of the present study was to further examine the relationship between serum insulin concentration and skeletal muscle carnitine accumulation by attempting to identify the serum insulin concentration at which this stimulatory effect of insulin on carnitine retention becomes apparent. On four randomized experimental visits, eight healthy men (body mass index 23.8 ± 0.9 kg/m2) underwent a 6-h euglycemic insulin clamp of 5, 30, 55, or 105 mU·m−2·min−1 accompanied by a 5-h iv infusion of l-carnitine (15 mg/kg bolus followed by 10 mg·kg−1·h−1). The clamps produced steady-state serum insulin concentrations of 10.1 ± 0.5, 48.8 ± 1.0, 88.9 ± 2.8, and 173.9 ± 6.5 mU/l, respectively. During l-carnitine infusion, plasma TC concentration remained above 450 μmol/l during all four visits. However, there was a significant treatment effect of insulin ( P < 0.001), such that by the end of infusion the plasma TC concentration in the 55- and 105-mU clamps was lower than that seen in the 5- ( P < 0.05 and P < 0.01, respectively) and 30-mU ( P < 0.01) clamps. The findings demonstrate that only high circulating serum insulin concentrations (≥90 mU/l) are capable of stimulating skeletal muscle carnitine accumulation. This is of relevance to athletes, and the treatment of obesity and type 2 diabetes, where increasing skeletal muscle carnitine content may be used as tool to modify skeletal muscle energy metabolism.