Dietary copper-fructose interactions contribute to the development of nonalcoholic fatty liver disease (NAFLD). Gut microbiota play critical roles in the pathogenesis of NAFLD. The aim of this study was to determine the effect of different dietary doses of copper and their interactions with high fructose on gut microbiome. Male weanling Sprague-Dawley rats were fed diets with adequate copper (6 ppm, CuA), marginal copper (1.5 ppm, CuM) (low-copper) or supplemented copper (20 ppm, CuS) (high-copper) for 4 weeks. Deionized water or deionized water containing 30% fructose (w/v) was given ad lib. Copper status, liver enzymes, gut barrier function and gut microbiome were evaluated. Both low- and high-copper diets led to liver injury in high fructose fed rats, and this was associated with gut barrier dysfunction, as shown by the markedly decreased tight junction proteins and increased gut permeability. 16S rDNA sequencing analysis revealed distinct alterations of the gut microbiome associated with dietary low- and high-copper/high-fructose feeding. The common features of the alterations of the gut microbiome were the increased abundance of Firmicutes and the depletion of Akkermansia. However, they differed mainly within the phylum, Firmicutes. Our data demonstrated that a complex interplay between host, microbes and dietary copper-fructose interaction regulates gut microbial metabolic activity, which may contribute to the development of liver injury and hepatic steatosis. The distinct alterations of gut microbial activity, which were associated with the different dietary doses of copper and fructose, imply that separate mechanism(s) may be involved.