Insulin resistant type 2 diabetic patients have been reported to have impaired skeletal muscle mitochondrial respiratory function. A key question is whether decreased mitochondrial respiration contributes directly to the decreased insulin action. To address this, a model of impaired cellular respiratory function was established by incubating human skeletal muscle cell cultures with the mitochondrial inhibitor sodium azide and the effects on insulin action examined. Incubation of human skeletal muscle cells with 50µM and 75µM azide resulted in a 48 ± 3% and a 56 ±1% decrease in respiration compared to untreated cells mimicking the level of impairment seen in type 2 diabetes. Under conditions of decreased respiratory chain function, insulin-independent (basal) glucose uptake was significantly increased. Basal glucose uptake was 325 ± 39 pmol/min/mg (mean ± SEM) in untreated cells. This increased to 669 ± 69 and 823 ± 83 pmol/min/mg in cells treated with 50 and 75µM azide, respectively (vs untreated, both p<0.0001). Azide treatment was also accompanied by an increase in basal glycogen synthesis and phosphorylation of AMP-activated protein kinase (AMPK). However, there was no decrease in glucose uptake following insulin exposure, and insulin-stimulated phosphorylation of Akt was normal under these conditions. GLUT1 mRNA expression remained unchanged while GLUT4 mRNA expression increased following azide treatment. In conclusion, under conditions of impaired mitochondrial respiration, there was no evidence of impaired insulin signalling or glucose uptake following insulin exposure in this model system.