© 2017 by the American Association for the Study of Liver Diseases. Portal hypertension (PH) drives most of the clinical complications in chronic liver diseases. However, its progression in nonalcoholic steatohepatitis (NASH) and its association with the intestinal microbiota (IM) have been scarcely studied. Our aim was to investigate the role of the IM in the mechanisms leading to PH in early NASH. The experimental design was divided in two stages. In stage 1, Sprague-Dawley rats were fed for 8 weeks a high-fat, high-glucose/fructose diet (HFGFD) or a control diet/water (CD). Representative rats were selected as IM donors for stage 2. In stage 2, additional HFGFD and CD rats underwent intestinal decontamination, followed by IM transplantation with feces from opposite-diet donors (heterologous transplant) or autologous fecal transplant (as controls), generating four groups: CD-autotransplanted, CD-transplanted, HFGFD-autotransplanted, HFGFD-transplanted. After IM transplantation, the original diet was maintained for 12-14 days until death. HFGFD rats developed obesity, insulin resistance, NASH without fibrosis but with PH, intrahepatic endothelial dysfunction, and IM dysbiosis. In HFGFD rats, transplantation with feces from CD donors caused a significant reduction of PH to levels comparable to CD without significant changes in NASH histology. The reduction in PH was due to a 31% decrease of intrahepatic vascular resistance compared to the HFGFD-autotransplanted group (P < 0.05). This effect occurs through restoration of the sensitivity to insulin of the hepatic protein kinase B–dependent endothelial nitric oxide synthase signaling pathway. Conclusion: The IM exerts a direct influence in the development of PH in rats with diet-induced NASH and dysbiosis; PH, insulin resistance, and endothelial dysfunction revert when a healthy IM is restored. (Hepatology 2018;67:1485-1498).