Prolonged intense exercise is challenging for the liver to maintain plasma glucose levels. Hormonal changes cannot fully account for exercise-induced hepatic glucose production (HGP). Contracting skeletal muscles release interleukin-6 (IL-6), a cytokine able to increase endogenous glucose production during exercise. However, whether this is due to a direct effect of IL-6 on liver remains unknown. Here, we studied hepatic glycogen, gluconeogenic genes and IL-6 signaling in response to one bout of exhaustive running exercise in rats. To determine whether IL-6 can modulate gluconeogenic genes mRNA independently of exercise, we injected resting rats with recombinant IL-6. Exhaustive exercise resulted in a profound decrease in liver glycogen and an increase in gluconeogenic genes mRNA levels, phosphoenolpyruvate-carboxykinase (PEPCK), glucose-6-phosphatase (G6P) and peroxisome proliferator-activated receptor coactivator-1 (PGC-1), suggesting a key role for gluconeogenesis in hepatic glucose production. This was associated to an active IL-6 signaling in liver tissue, as shown by signal transducer and activator of transcription (STAT-3) and CAAT/enhancer binding protein- (C/EBP) phosphorylation and IL-6 responsive genes mRNA levels at the end of exercise. Recombinant IL-6 injection resulted in an increase in IL-6 responsive genes mRNA levels in the liver. We found a dose-dependent increase in PEPCK gene mRNA, strongly correlated with IL-6-induced genes mRNA levels. No changes in G6P and PGC-1 mRNA levels were found. Taken together, our results suggest that during very demanding exercise, muscle-derived IL-6 could help increasing HGP by directly up regulating PEPCK mRNA abundance.