• Lactobacillus -fermented black barley exhibited preventative effect against NAFLD. • It also exhibited regulatory effect on intestinal microbiota dysbiosis by increasing the microbiota relative abundance, diversity and enriching some intestinal probiotics; affected some of the fecal metabolites related to hormones and lipid metabolism. • Fermented black barley might act as an effective and safe preventive dietary supplementation against fatty liver induced by high fat diet. Gut microbiota dysbiosis and oxidative stress may play important roles in the progression of nonalcoholic fatty liver disease (NAFLD). Fermented foods contain probiotics and other bioactive components that may improve gastrointestinal health and provide other health benefits. Here, we investigated the effect of Lactobacillus -fermented black barley on NAFLD rats. Adult Sprague Dawley rats were randomized into four groups: normal chow diet (NC), high-fat diet (HF), HF + fermented black barley treatment (HB) and HF + Lactobacillus treatment (HL). The rats in the HB and HL groups were continuously administered fermented black barley or Lactobacillus , respectively, for 12 weeks (1 mL/100 g·BW, containing 1 × 10 8 CFU/mL Lactobacillus ). Compared with the HF treatment, HB treatment effectively inhibited the increase in body weight, liver and abdominal fat indexes and hepatic lipids ( p < 0.01), increased hepatic SOD activity ( p < 0.05), decreased thiobarbituric acid reactive substances (TBARSs) ( p < 0.01) and improved liver function. Moreover, Lactobacillus -fermented black barley exhibited regulatory effect on high-fat diet-induced intestinal microbiota dysbiosis by increasing the relative microbiota abundance and diversity, increasing the relative abundance of Bacteroidetes , decreasing the Firmicutes/Bacteroidetes ratio, increasing the abundances of some intestinal probiotics (such as Akkermansia and Lactococcus ), and influencing some of the fecal metabolites related to hormones and lipid metabolism. The supplementation of fermented cereal food might be a new effective and safe preventive dietary strategy against NAFLD.