The ability to maintain skeletal muscle mass appears to be impaired in insulin-resistant conditions, such as type 2 diabetes, that are characterized by muscle lipid accumulation. The current study investigated the effect of acutely increasing lipid availability on muscle protein synthesis. Seven healthy young male volunteers underwent a 7-h intravenous infusion of l-[ring-2H5]phenylalanine on two randomized occasions combined with 0.9% saline or 10% Intralipid at 100 mL/h. After a 4-h “basal” period, a 21-g bolus of amino acids was administered and a 3-h hyperinsulinemic-euglycemic clamp was commenced (“fed” period). Muscle biopsy specimens were obtained from the vastus lateralis at 1.5, 4, and 7 h. Lipid infusion reduced fed whole-body glucose disposal by 20%. Furthermore, whereas the mixed muscle fractional synthetic rate increased from the basal to the fed period during saline infusion by 2.2-fold, no change occurred during lipid infusion, despite similar circulating insulin and leucine concentrations. This “anabolic resistance” to insulin and amino acids with lipid infusion was associated with a complete suppression of muscle 4E-BP1 phosphorylation. We propose that increased muscle lipid availability may contribute to anabolic resistance in insulin-resistant conditions by impairing translation initiation.