Di-(2-ethyl hexyl) phthalate (DEHP) is the plasticizer used in variety of medical and consumer products to impart structural flexibility. DEHP and its primary metabolite mono-(2-ethyl hexyl)phthalate (MEHP) posed a considerable interest because of their contribution to insulin resistance, type-2 diabetes and obesity. Experimental and epidemiological data have shown that DEHP affects blood glucose homeostasis. However, direct effect of DEHP and its metabolite MEHP on insulin signal transduction and glucose transporter 4 (GLUT4) translocation remain obscure. The present study was delineated to decipher the direct effects of DEHP and MEHP on insulin signal transduction and proteins involved in GLUT4 translocation in cultured L6 myotubes, the rat skeletal muscle model. For this study we have exposed cells with 50 and 100μM DEHP and MEHP for 24h followed by insulin stimulation for 20min. GLUT4 level in both cytosol and plasma membrane fractions were analysed by western blot analysis and found to be significantly decreased. Further, DEHP and MEHP treatment significantly altered the insulin signalling molecules and proteins involved in GLUT4 translocation (Rab8A (Ras related proteins in skeletal muscle), insulin - regulated amino peptidase (IRAP), synaptosomal - associated protein 23 (SNAP23), Syntaxin4, Munc18c) from cytosol to plasma membrane. Impaired GLUT4 in the plasma membrane resulted in decreased 14C-deoxy glucose uptake. 14C-glucose oxidation and glycogen content were also significantly decreased in treated groups. In essence, the present study is first of its kind to show the direct adverse effects of DEHP and MEHP on insulin signal transduction and GLUT4 translocation in cultured L6 myotubes. Further, MEHP is found to be more effective than DEHP as a result of its differential structure and physico-chemical properties.