Purpose: To investigate the mechanisms of impairments in oxidative metabolism in obese and diabetic (T2DM) skeletal muscle, this study analysed the adaptive expression of genes involved in fatty acid (FA) oxidation and mitochondrial biogenesis in primary myotubes treated with elevated FAs. Methods: Muscle samples from obese or obese T2DM donors were stored or processed into human primary skeletal muscle myotubes, which were treated for 6 h with a saturated (palmitic acid) or a monounsaturated (oleic acid) FA with or without a polyunsaturated FA (eicosapentaenoic acid: EPA). Real-time PCR analysis was used to determine mRNA expression. Results: Basal pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in whole muscle samples from obese and T2DM subjects was increased compared to lean (P <0.05; n = 13?20/group). In obese- and T2DM-derived myotubes, oleic acid treatment alone and in combination with EPA increased PDK4 mRNA expression compared to control (P <0.05; n = 7/group), whereas palmitic acid alone and in combination with EPA only increased PDK4 mRNA in T2DM-derived myotubes compared to control (P <0.05; n = 7/group). EPA alone did not alter mRNA expression of PDK4. Conclusions: These findings show that FAs induce the expression of PDK4 mRNA, which was increased in myotubes cultured from obese and T2DM donors. This persistent difference in PDK4 expression, present after culturing, suggests a fundamental alteration in the FA-mediated gene expression. This may in turn translate to differences in the regulation of oxidative substrate flux to impact on insulin sensitivity.