Sterol Regulatory Element Binding Protein-1c (SREBP-1c) is a transcription factor that regulates genes associated with glucose and fat metabolism (eg HKII, ACC and FAS). Animal studies have shown SREBP-1c to exhibit responsiveness to insulin and exercise. However, the signalling pathways regulating SREBP1c in human skeletal muscle are not clear. The Akt and the p38 mitogen-activated protein kinase (MAPK) pathways have been suggested as positive regulators whereas the STAT3 and extracellular signal-regulated kinase (ERK 1/2) pathways have been suggested as negative regulators of SREBP1c expression. The aim of this study was to examine the effects of exercise on basal and insulin-mediated changes in the activation (phosphorylation) of these signalling molecules and relate them to changes in SREBP-1c expression in human skeletal muscle. Eight healthy men (age 24 ± 2yrs, BMI 24 ± 1 kg/m2, mean ± SEM) underwent a hyperinsulinaemic (80 mU/l) euglycaemic (4.5 mmol/l) clamp for 4h, 24h after 90min of one-legged cycling at moderate intensity while keeping the other leg rested (control leg). During the subsequent 24h recovery, subjects consumed a mixed diet (55% CHO, 30% fat). Muscle biopsy samples were obtained from both legs after exercise (day 1), and before and after the insulin clamp (Day 2). Immediately after exercise, phosphorylation of pERK1 (0.41 ± 0.08 vs 0.32 ± 0.05, P<0.05, 1-way ANOVA), pERK2 (0.52 ± 0.10 vs 0.26 ± 0.05, P<0.05) and pAkt (0.78 ± 0.07 vs 0.63 ± 0.06, P<0.05) was higher in the exercise than the control leg, respectively. SREBP-1c mRNA content was not affected by exercise whereas its protein level was lower in the exercise than the control leg immediately after exercise (1.57 ± 0.51 vs 3.58 ± 0.99, P<0.05) and returned to normal levels 24h later. Similarly, SREBP-1c mRNA content was ~1.5 fold higher (P<0.01) in the exercise than the control leg 24h later. On day 2, insulin infusion upregulated SREBP1c mRNA levels by ~2-fold (P<0.01) in both legs but did not affect its protein levels. Although there were no insulin-induced changes in pERK1/2, insulin infusion increased pAkt levels in the control leg (0.97 ± 0.11 vs 0.66 ± 0.06, P<0.05) and tended to increase it in the exercise leg (0.90 ± 0.06 vs 0.63 ± 0.05, P=0.065). Neither exercise nor insulin affected STAT3 or p38 MAPK phosphorylation. These findings suggest that the exercise-induced reduction in skeletal muscle SREBP1c expression might be mediated by increases in the pERK1/2 pathway, whereas Akt might be an important positive regulator of SREBP1c mRNA in human skeletal muscle under insulin-stimulated conditions.