Exercise training is commonly prescribed for treatment of non-alcoholic fatty liver disease (NAFLD). We sought to determine whether exercise training prevents the development of NAFLD in Otsuka Long-Evans Tokushima Fatty (OLETF) rats and to elucidate the molecular mechanisms underlying the effects of exercise on hepatic steatosis. Four-week old OLETF rats were randomly assigned to either a sedentary control group (SED) or given access to voluntary running wheels for 16 weeks (EX). Wheels were locked two days before sacrifice in the EX animals, and both groups were sacrificed at 20 weeks old. Voluntary wheel running attenuated weight gain and reduced serum glucose, insulin, free fatty acids, and triglycerides in EX animals compared with SED (p<0.001). EX animals exhibited significantly reduced hepatic triglyceride levels, displayed fewer lipid droplets (Oil-Red O staining), and reduced lipid droplet size compared to SED. Wheel running increased by 3-fold the percent of palmitate oxidized completely to CO₂ in the EX animals, but did not alter AMP-activated protein kinase- (AMPK) or AMPK phosphorylation status. However, fatty acid synthase (FAS) and acetyl-coenzyme A carboxylase (ACC) content were significantly reduced (~70% and ~35%, respectively), and ACC phosphorylation and cytochrome c content were significantly elevated (~35% and ~30%, respectively) in the EX animals. These results unequivocally demonstrate that daily physical activity attenuates hepatic steatosis and NAFLD in an obese rodent model and suggest that this effect is likely mediated, in part, through enhancement of hepatic fatty acid oxidation and reductions in key protein intermediates of fatty acid synthesis.