ABSTRACT: Six steers were used to study the effects of dietary supplementation with sodium sulfate (Na2SO4) on rumen fermentation, nutrient digestion, rumen microbiota, and plasma metabolites. The animals were fed a basal ration with Na2SO4 added at 0 g/day (sulfur [S] content of 0.115% dry matter [DM]), 20 g/day (S at 0.185% DM), or 40 g/day (S at 0.255% DM) in a replicate 3-by-3 Latin square design. The results indicated that supplementing with Na2SO4 increased the ruminal concentration of total volatile fatty acids, the molar proportions of acetate and butyrate, the ruminal concentrations of microbial protein, SO4 2--S, and S2--S, and the digestibility of fiber, while it decreased the molar proportion of propionate and the ruminal concentration of ammonia nitrogen. Supplementing with Na2SO4 increased the diversity and the richness of rumen microbiota and the relative abundances of the phylum Firmicutes and genera Ruminococcus 2, Rikenellaceae RC9 gut group, and Desulfovibrio, whereas it decreased the relative abundances of the phylum Bacteroidetes and genera Prevotella 1, Prevotellaceae UCG-001, and Treponema 2 Supplementing with Na2SO4 also increased the plasma concentrations of amino acids (l-arginine, l-methionine, l-cysteine, and l-lysine), purine derivatives (xanthine and hypoxanthine), vitamins (thiamine and biotin), and lipids (acetylcarnitine and l-carnitine). It was concluded that supplementing the steer ration with Na2SO4 was beneficial for improving the rumen fermentation, fiber digestibility, and nutrient metabolism through modulating the rumen microbial community.IMPORTANCE Essential elements like nitrogen and sulfur greatly affect rumen fermentation and metabolism in ruminants. However, little knowledge is available on the effects of sulfur on the rumen microbiota and plasma metabolome. The results of the present trial demonstrated that supplementing the steer ration with sodium sulfate markedly improved rumen fermentation, fiber digestibility, and metabolism of amino acids, purine derivatives, and vitamins through effects on the ruminal microbiome. The facts obtained from the present trial clarified the possible mechanisms of the positive effects of sulfur on rumen fermentation and nutrient utilization.