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Gut microbes, especially those in the large intestine, are actively involved in nutrient metabolism; however, their impact on host nitrogen (N) metabolism remains largely unknown. This study was designed to investigate the effects of feeding a cocktail of antibiotics (AGM) (ampicillin, gentamycin and metronidazole) on intestinal microbiota, N utilization efficiency, and amino acid (AA) digestibility in cannulated pigs, with the aim of exploring the impact of gut microbiota on host N metabolism. In total, 16 piglets were surgically fitted with a simple distal ileal T-cannula and a jugular venous catheter. The pigs were fed a basal diet without antibiotics (control; CON) or with antibiotics (antibiotic; ANTI), for 2 weeks. The results showed that feeding AGM did not affect weight gain or digestive enzyme activity. The antibiotics increased the concentration of urea N (P <0.05). However, they reduced N utilization, and the total tract apparent digestibility of isoleucine, methionine, valine, tyrosine and total AA (P <0.05). Furthermore, the antibiotics increased the terminal ileum apparent digestibility of CP, phenylalanine, valine, alanine, tyrosine and total AA (P <0.05). AGM markedly altered the composition of the microbiota in the ileum and feces, with a reduction in populations of Bifidobacterium , Lactobacillus and Ruminococcus , and an increase in the abundance of Escherichia coli (P <0.05). The antibiotics also significantly increased the concentration of cadaverine and ammonia, both in ileal digesta and feces (P <0.05), suggesting a marked impact on N metabolism in the intestine. The analyses indicated that the alteration of gut microbiota was correlated with the apparent digestibility of CP and AA in the intestine. These findings suggest that the AGM-induced alteration of gut microbiota may contribute to the change in intestinal N metabolism, and consequently, N excretion from the body. These results also suggest that antibiotics could have a significant effect on host N metabolism. The present study contributes to our understanding of the effects of antibiotics and provides a rational scientific basis for diet formulation during AGM use. [ABSTRACT FROM AUTHOR]