In the present study, we investigated the influence of Lactobacillus plantarum and Lactobacillus helveticus supplementation on lipogenesis, insulin signalling and glucose transporters in liver of high-fructose-fed rats. Fructose was given to the rats as a 20% solution in drinking water for 15 weeks. Lactobacillus plantarum and L. helveticus supplementations were performed by gastric gavage once a day during final 6 weeks. Dietary high-fructose increased hepatic weight, lipid accumulation and FASN expression as well as caused a significant reduction in IRS-1 expression, pAKT/total AKT and peNOS/total eNOS ratios, but an elevation in GLUT2 and GLUT5 mRNAs in the liver. Lactobacillus plantarum supplementation decreased hepatic weight, triglyceride content and FASN expression as well as improved IRS-1/AKT/eNOS pathway and GLUT2 expression in the liver of high-fructose-fed rats. However, L. helveticus supplementation exerted a restoring effect on lipid accumulation by decreasing FASN expression, and regulating effect on IRS-1 and GLUT2 expressions.