Clinical studies have linked the increased consumption of fructose to the development of obesity, dyslipidemia and impaired glucose tolerance, and a role in hepatosteatosis development is presumed. Fructose can undergo a non-enzymatic reaction from which advanced glycation endproducts (AGEs) are derived, leading to the formation of dysfunctional, fructosylated proteins, however the in vivo formation of AGEs from fructose is still less known than that from glucose. In the present study C57Bl/6J mice received 15% (w/v) fructose (FRT) or 15% (w/v) glucose (GLC) in water to drink for 30 weeks, resembling human habit to consume sugary drinks. At the end of protocol both FRT and GLC drinking mice had increased fasting glycaemia, glucose intolerance, altered plasma lipid profile, and marked hepatosteatosis. FRT mice had higher hepatic triglycerides deposition than GLC, paralleled by a greater increased expression and activity of the sterol regulatory element-binding protein 1 (SREBP1), the transcription factor responsible for the de novo lipogenesis, and of its activating protein SCAP. LC-MS analysis showed a different pattern of AGEs production in liver tissue between FRT and GLC mice, with larger amount of carboxymethyl lysine (CML) generated by FRT. Double immunofluorescence and coimmunoprecipitation analysis revealed an interaction between CML and SCAP that could lead to prolonged activation of SREBP1. Overall, the high levels of CML and activation of SCAP/SREBP pathway associated to high fructose exposure here reported may suggest a key role of this signaling pathway in mediating fructose-induced lipogenesis.