To test the hypothesis that metformin protects against fructose-induced steatosis, and if so, to elucidate underlying mechanisms, C57BL/6J mice were either fed 30% fructose solution or plain water for 8 weeks. Some of the animals were concomitantly treated with metformin (300 mg/kg body weight/day) in the drinking solution. While chronic consumption of 30% fructose solution caused a significant increase in hepatic triglyceride accumulation and plasma alanine-aminotransferase levels, this effect of fructose was markedly attenuated in fructose-fed mice concomitantly treatment with metformin. The protective effects of the metformin treatment on the onset of fructose-induced non-alcoholic fatty liver disease (NAFLD) were associated with a protection against the loss of the tight junction proteins occludin and zonula occludens 1 in the duodenum of fructose-fed mice and the increased translocation of bacterial endotoxin found in mice only fed with fructose. In line with these findings, in metformin-treated fructose-fed animals, hepatic expression of genes of the toll-like receptor-4-dependent signalling cascade as well as the plasminogen-activator inhibitor/cMet-regulated lipid export were almost at the level of controls. Taken together, these data suggest that metformin not only protects the liver from the onset of fructose-induced NAFLD through mechanisms involving its direct effects on hepatic insulin signalling but rather through altering intestinal permeability and subsequently the endotoxin-dependent activation of hepatic Kupffer cells.