β-secretase (β-site APP Cleaving Enzyme 1 (BACE1)) is a key player in the development and progression of Alzheimer’s disease (AD). Sequential cleavage of amyloid precursor protein by β- and γ-secretase produces β-amyloid peptides, the major component of plaques in AD patient brains. BACE1 has been validated as a therapeutic target for AD as BACE1 knockout (KO) mice have low β-amyloid levels. BACE1 KO mice also exhibit a lower body weight and better glucose disposal than their wild type littermates. Using C2C12 (mouse) and L6 (rat) cultured myoblasts we show that pharmacological inhibition of BACE1 enhances insulin signalling, as measured by protein kinase B phosphorylation (p-PKB). Treatment of C2C12 cells with 5 nM insulin increased p-PKB levels by 6.07 ± 1.27 fold (P < 0.001, n = 10), compared to un-stimulated cells. A 24-hour pre-treatment with 250 nM BACE1 inhibitor (1) enhanced insulin-stimulated p-PKB levels, 10.27 ± 1.52 fold (P < 0.001, unpaired Student’s t-test, mean ± SEM; n = 10) compared to control cells. Comparison of insulin-stimulated p-PKB levels in the presence and absence of the BACE1 inhibitor showed that BACE1 inhibition significantly enhanced insulin stimulated p-PKB levels (P < 0.05). L6 myoblasts were more insulin sensitive, with 0.3 nM insulin increasing p-PKB levels by 3.18 ± 0.55 fold (P < 0.01, n = 4) compared to control cells. L6 myoblasts pre-treated with BACE1 inhibitor increased p-PKB by 5.15 ± 0.79 fold in response to insulin, (P < 0.001 versus control; n = 4) and a significant enhancement in insulin sensitivity (P < 0.05). In cultured L6-GLUT4myc cells, treatment with BACE1 inhibitor per se increased translocation of the glucose transporter (GLUT4; 1.53 ± 0.08 fold; P < 0.001, n = 5). Insulin (100 nM) increased GLUT4 translocation by 1.40 ± 0.11 fold (P < 0.01, n = 5) in untreated cells and by 1.82 ± 0.20 fold (P < 0.05, n = 5) in cells pre-treated with BACE1 inhibitor. Thus, BACE1 inhibition increases GLUT4 translocation supporting the notion that BACE1 reduction enhances glucose uptake. However, at least part of this effect in these muscle cells appears insulin-independent. Raised 5’ AMP activated protein kinase (AMPK) activity increases GLUT4 translocation independently of insulin (2). Treatment of C2C12 and L6 cells with BACE1 inhibitor increased AMPK phosphorylation (1.66 ± 0.43 fold; P = 0.06, n = 8 and 1.72 ± 0.39 fold; P < 0.05, n = 11 respectively) and phosphorylation of its substrate, acetyl CoA carboxylase (ACC; 1.72 ± 0.17 fold; P < 0.01, n = 8 and 1.33 ± 0.21 fold; P = 0.06, n = 11 respectively), indicative of enhanced AMPK activity. Thus, we propose that skeletal muscle BACE1 levels modify glucose uptake in an insulin and AMPK dependent manner.