The aim of the present study was to examine whether pretreatment with different fatty acids (FAs), as well as the LXR agonist T0901317, could modify metabolic switching of human myotubes. The n-3 FA eicosapentaenoic acid (EPA) increased suppressibility, the ability of glucose to suppress FA oxidation. Substrate-regulated flexibility, the ability to increase FA oxidation when changing from a high glucose, low fatty acid condition (''fed'') to a high fatty acid, low glucose (''fasted'') condition, was increased by EPA and other n-3 FAs. Adaptability, the capacity to increase FA oxidation with increasing FA availability, was enhanced after pretreatment with EPA, linoleic acid (LA) and palmitic acid (PA). T0901317 counteracted the effect of EPA on suppressibility and adaptability, but did not affect these parameters alone. EPA per se accumulated less, however, EPA, LA, oleic acid and T0901317-treatment increased the number of lipid droplets (LDs) in myotubes. LD volume and intensity, as well as mitochondrial mass were independent of FA pretreatment. Microarray analysis showed that EPA regulated more genes than the other FAs and specific pathways involved in carbohydrate metabolism were induced only by EPA. The present study suggests a favorable effect of n-3 FAs on skeletal muscle metabolic switching and glucose utilization.