Fasting prompts a metabolic shift in substrate utilization from carbohydrate to predominant fat oxidation in skeletal muscle and pyruvate dehydrogenase (PDH) is seen as a controlling link between the competitive oxidation of carbohydrate and fat during metabolic challenges like fasting. Interleukin (IL)-6 has been proposed to be released from muscle with concomitant effects on both glucose and fat utilization. The aim was to test the hypothesis that IL-6 has a regulatory impact on fasting-induced suppression of skeletal muscle PDH. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (Control) were either fed or fasted for 6 or 18 hours. Lack of muscle IL-6 elevated the respiratory exchange ratio (RER) in the fed and early fasting state, but not with prolonged fasting. PDHa activity was higher in fed and fasted IL-6 MKO than Control mice, while lack of muscle IL-6 did not prevent down-regulation of PDHa activity in skeletal muscle or changes in plasma and muscle substrate levels in response to 18h of fasting. Phosphorylation of 3 out of 4 sites on PDH-E1α increased with 18h of fasting, but was lower in IL-6 MKO mice than Control. In addition, both PDK4 mRNA and protein increased with 6h and 18h of fasting in both genotypes, but PDK4 protein was lower in IL-6 MKO than Control. In conclusion, muscle IL-6 seems to regulate resting substrate utilization potentially through effects on skeletal muscle PDH regulation, but is not required for maintaining metabolic flexibility in response to fasting.