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Glucose homeostasis depends on insulin-regulated glucose uptake in the skeletal muscles and fat tissues via glucose transporter (GLUT) 4 translocation into cellular plasma membranes. The present study sought to elucidate GLUT4 expression, GLUT1 and GLUT4 translocation and glucose uptake in the skeletal muscles of Shetland ponies. Semitendinosus muscle explants were removed by open muscle biopsy from six Shetland pony geldings under general anaesthesia. The expression of GLUT4 was analysed by measuring muscle crude membrane (CM) GLUT4 protein contents. To determine the insulin-stimulated GLUT translocation, GLUT1 and GLUT4 concentrations were measured in partially purified plasma membranes (PM) and cytoplasmic vesicles (CV). GLUT contents were determined semi-quantitatively by Western blotting. Insulin-stimulated glucose uptake was analysed using 3-O-d-methyl[(3)H]glucose uptake. Incubation of semitendinosus muscle strips with 0.1 and 20mIU/mL insulin significantly increased GLUT4 translocation (PM GLUT4 contents), but had no significant effect on GLUT4 expression (CM GLUT4 concentrations) or PM GLUT1. The uptake of myocyte 3-O-Methylglucose was not significantly increased following insulin stimulation. The sub-cellular fractionation technique proved to be an appropriate tool for determining insulin-stimulated GLUT4 translocation in equine skeletal muscle. GLUT4 translocation in equines is insulin-dependent, as has been described in rodents and farm animals, but insulin-stimulated GLUT4 activation in ponies is lower than reported for pigs and cows under the same experimental conditions. Poor insulin-activated GLUT4 translocation may account for insulin resistance in ponies in previous euglycaemic, hyperinsulinaemic clamp tests.