The endocannabinoids, a recently discovered endogenous, lipid derived, signaling system regulating energy metabolism, have effects on central and peripheral energy metabolism predominantly via the cannabinoid receptor type 1 (CB1). CB1 is expressed centrally in the hypothalamus and nucleus accumbens and peripherally in adipocytes and skeletal muscle. This study determined the effect of endocannabinoids on the expression of genes regulating energy metabolism in human skeletal muscle. Primary cultures of myotubes (lean and obese; n=3/group) were treated with the cannabinoid receptor agonist, anandamide (AEA) (0.2 and 5μM) and the CB1 specific antagonist AM251 (0.2 and 5μM) separately and in combination for 24h. The expression of mRNA for AMP-activated protein kinase (AMPK) alpha 1 (α1) and alpha 2 (α2), pyruvate dehydrogenase kinase 4 (PDK4) and peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) were determined using ‘Real Time’ RT-PCR. AMPKα1 mRNA increased in lean and obese myotubes in response to AM251 (P<0.05). AEA inhibited the effect of AM251 on AMPKα1 mRNA levels in myotubes from lean and obese subjects (P<0.05); the dose–response curve was shifted to the left in the obese. In response to AM251, irrespective of the presence of AEA, PDK4 expression was decreased in lean and obese myotubes (P<0.05). Taken together these data suggest that endocannabinoids regulate pathways affecting skeletal muscle oxidation, effects particularly evident in myotubes from obese individuals.