Renal tubular lipid accumulation is associated with renal injury in the metabolic syndrome, but its mechanisms are not fully elucidated. The purpose of the present study was to investigate the exact mechanism of renal tubular lipid accumulation in the diet-induced metabolic syndrome. The in vivo experiments showed that a high-fat diet induced hyperglycaemia, hyperinsulinaemia and hypertriacylglycerolaemia, subsequent increases in sterol regulatory element binding protein-1 (SREBP-1) and transforming growth factor-β1 (TGF-β1), lipid droplet deposit in renal tubular cells and interstitial extracellular matrix accumulation in Wistar rats. A human renal proximal tubular epithelial cell line (HKC) was used to determine the direct role of insulin, and the results revealed that insulin induced SREBP-1, fatty acid synthase (FASN), TGF-β1 expressions, lipid droplet and extracellular matrix deposits. Knockdown of SREBP-1 by RNA interference technology significantly inhibited FASN, TGF-β1 up-regulation, lipid and extracellular matrix accumulation caused by insulin. In addition, we found that insulin and high glucose could synergistically increase SREBP-1, FASN, TGF-β1 and fibronectin expressions in HKC cells. These results indicate that high-fat diet-induced increased serum insulin and glucose synergistically cause renal tubular lipid deposit and extracellular matrix accumulation via the SREBP-1 pathway.