Insulin is known to inhibit muscle protein breakdown in humans (Bennett & Rennie, 1991), but the mechanism by which this is achieved is unknown. Skeletal muscle proteins are predominantly degraded via the ubiquitin-proteasome system. Of particular note, the muscle specific ubiquitin E3 ligases, muscle-specific muscle ring finger 1 (MuRF1) and muscle atrophy box factor (MAFbx), have been shown to be markedly up-regulated in animal models and human wasting conditions in multiple catabolic states, such as sepsis, renal failure and burns (Wray et al. 2003; Lecker et al. 2004). The aim of this study was to determine whether insulin-mediated inhibition of muscle protein breakdown is associated with a concomitant down-regulation of MAFbx mRNA and protein expression in human skeletal muscle, and whether this response is dose dependent. Following an overnight fast, 8 healthy young men (20.4 ± 1.2 y, BMI 23.8 ± 2.6 kg/m²) had leg protein breakdown determined, using D5-phenylalanine, before and during a hyperinsulinaemic-euglycaemic clamp, during which mixed amino acid infusion also occurred (18 g/h, Glamin, Kabi Fresenius). Leg protein breakdown was measured before and during 3 h of insulin infusion on 4 separate occasions with the aim of maintaining steady-state plasma insulin concentrations of ~5 (achieved with octreotide (30 ng/kg/min with replacement glucagon (15 ng/kg/h) and 20% glucose), ~30, ~80 and ~170 mU/l. Needle biopsy samples were obtained from the quadriceps muscle in the basal state and after 3 h infusion to determine MAFbx mRNA and protein expression. Infusion of amino acids when insulin was maintained at ~5 mU/l (equivalent to the fasted state) had no effect on leg protein breakdown. However, when insulin availability was increased to ~30 mU/l protein breakdown was suppressed by ~50% (P<0.001), and was maintained at this rate as insulin availability was increased further. MAFbx mRNA expression was unchanged throughout the study. Infusion of amino acids, when insulin was maintained at ~5 mU/l, reduced MAFbx protein expression by 47% from basal (P<0.01). Increasing steady-state plasma insulin concentration to ~30, ~80 and ~170 mU/l was accompanied by a decline in MAFbx protein expression from basal by 80% (P<0.01), 87% (P<0.01) and 90% (P<0.01), respectively. This study demonstrates, for the first time, that the inhibitory effect of insulin on muscle protein breakdown may be mediated via inhibition of MAFbx protein expression, although this relationship does not appear to happen in a dose-dependant manner. The inhibitory effect of amino acids on MAFbx protein expression warrants further investigation.