Transcriptional repressor hairy and enhancer of split 1 (HES1) is a basic helix-loop-helix DNA binding protein, and a downstream effector of Notch signalling. Hes1 has been metabolically implicated in adipose tissue, whereby overexpression inhibits the transcription of adipocyte-browning genes, Ppargc1a and Prdm16, through direct binding of their promoters (1). In skeletal muscle, Hes1 negatively regulates myogeneic differentiation by suppressing MyoD (2). Intriguingly, HES1 has been identified as an insulin-responsive gene in human vastus lateralis (3), and is less abundant in the proteome of obese, type 2 diabetic individuals (4). We aimed to elucidate the metabolic role of HES1 in human skeletal muscle. High-throughput analysis of the skeletal muscle transcriptome identified differential expression of HES1 between men with normal glucose tolerance (NGT; n=25, age=58±9 years) and men with type 2 diabetes (T2D; n=25, age=62±6 years): after a 2-hour oral glucose challenge, HES1 increased in males with NGT and T2D (P<0.0001, Wilcoxon sign test), however the extent of this increase was attenuated in those with T2D (3-fold, 95%CI [2.4, 3.8] vs 1.7-fold, 95%CI [1.4, 2.1]; P<0.0001, Mann-Whitney U test). Primary human skeletal muscle myotubes (pHSkMM) were used to further characterise HES1. Insulin treatment (120 nM; 30 minutes) elevated HES1 levels in pHSkMM (1.6-fold, IQR [1.2, 4.9]; P=0.06, Wilcoxon sign test; n=5). HES1 siRNA silencing (30%) altered the expression of glucose and fatty acid metabolism-regulating genes: GLUT1 (0.7±0.2-fold), HK2 (0.8±0.1-fold) and CPT1B (1.3±0.1-fold) (P<0.05, paired samples t-test vs scramble control; n=5). Furthermore, insulin-stimulated (10 and 120 nM) D-[14C (U)]-glucose incorporation into glycogen was attenuated in HES1-silenced myotubes, compared to scramble control (1.5±0.2-fold vs 0.9±0.1-fold and 1.9±0.2-fold vs 1.7±0.2-fold, respectively; P<0.05, RM 2-Way ANOVA with Šidák correction; n=5). Electrical pulse stimulation (3 hours) increased HES1 abundance in pHSkMM (1.8±0.3-fold; P=0.03, paired samples t-test; n=7). Moreover, cycling exercise (30 minutes at 85% of maximum heart rate) increased HES1 expression in skeletal muscle of men with NGT (n=24, age 61±6 years; VO2 max 35.5±7.6 mL.Kg.min-1) and T2D (n=26, age 60±5 years; VO2 max 31.4±7.6 mL.Kg.min-1) (P<0.0001; paired samples t-test). In summary, HES1 is blunted in the skeletal muscle of men with T2D after an oral glucose tolerance test. HES1 silencing modulates the expression of key metabolic genes and recapitulates an insulin-resistant phenotype in skeletal muscle, as indicated by reduced incorporation of glucose into glycogen with insulin stimulation. HES1 is increased by skeletal muscle contraction and responds similarly in NGT and T2D individuals after exercise. Our results implicate HES1 in the development of skeletal muscle insulin resistance and tentatively suggest that physical activity could restore HES1 levels in T2D.