Little was known regarding the differences in the gut microbiomes of brucellosis patients and healthy subjects (HCs). To explore the characteristics of the gut microbiota in brucellosis patients, 16s rRNA sequencing data from 40 Chinese HCs, 16 acute and 3 post-treatment brucellosis were analyzed. Acute and post-treatment brucellosis were lower in community richness and microbial diversity compared to HCs (P<0.05). Actinobacteria and subordinate genera, Bifidobacterium and Collinsella, were higher in acute brucellosis. Antibiotic treatment further reduced microbial diversity and enriched Gram-positive bacteria such as Enterococcus, Corynebactereraceae and Lactobacillales. PICRUSt2 analysis identified significant differences in 18 KEGG pathways, among which 10/13 (76.9%) metabolism pathway increased in brucellosis groups compared to HCs, indicating metabolic enhancement after Brucella infection. In conclusion, our cohort study detected altered microbiota and decreased microbial diversity in the brucellosis patients, marked by a significant overgrowth of disease-related microbes from Actinobacteria, which was a new perspective for the pathogenesis of brucellosis.