The liver is essential in maintaining and regulating glucose homeostasis during prolonged exercise. IL-6 has been shown to be secreted from skeletal muscle during exercise and has been suggested to signal to the liver. Therefore, the aim of this study was to investigate the role of skeletal muscle IL-6 on hepatic glucose regulation and substrate choice during prolonged exercise. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice (age, 12-14 wk) and littermate lox/lox (Control) mice were either rested (Rest) or completed a single bout of exercise for 10, 60, or 120 min, and the liver was quickly obtained. Hepatic IL-6 mRNA was higher at 60 min of exercise, and hepatic signal transducer and activator of transcription 3 was higher at 120 min of exercise than at rest in both genotypes. Hepatic glycogen was higher in IL-6 MKO mice than control mice at rest, but decreased similarly during exercise in the two genotypes, and hepatic glucose content was lower in IL-6 MKO than control mice at 120 min of exercise. Hepatic phosphoenolpyruvate carboxykinase mRNA and protein increased in both genotypes at 120 min of exercise, whereas hepatic glucose 6 phosphatase protein remained unchanged. Furthermore, IL-6 MKO mice had higher hepatic pyruvate dehydrogenase (PDH)^Ser232 and PDH^Ser300 phosphorylation than control mice at rest. In conclusion, hepatic gluconeogenic capacity in mice is increased during prolonged exercise independent of muscle IL-6. Furthermore, Skeletal muscle IL-6 influences hepatic substrate regulation at rest and hepatic glucose metabolism during prolonged exercise, seemingly independent of IL-6 signaling in the liver.