The mechanisms by which antibiotic growth promoters (AGP) enhance growth rates, feed efficiencies, and disease resistance in poultry need to be understood for designing safer and alternative strategies to replace AGP. Avilamycin has been widely used as an AGP in poultry, but its impact on the structure and function of the gut microbiome of broiler chickens has not been fully elucidated. In this study, we investigated the bacterial communities of the ileum and cecum in broiler chickens fed with an avilamycin-supplemented diet, by high-throughput sequencing of bacterial 16S rRNA genes. Alpha diversity metrics indicated that the ileal bacterial diversity was higher in avilamycin-fed chickens than in the control group, whereas the opposite was true for the cecum. Multivariate analyses revealed that the ileal microbiota of the avilamycin-fed group were clearly distinguished from those of the control group, whereas the cecal bacterial communities were apparently not influenced by feeding diets containing avilamycin. In the ilea, 2 operational taxonomic units (OTU) that matched Lactobacillus reuteri and Clostridium were enriched (P = 0.016 and P = 0.007, respectively) in the avilamycin-fed group, and an OTU belonging to Lactobacillus crispatus was decreased (P = 0.016). In the cecal microbiota showing much higher diversity with 1,286 non-singleton OTU, 12 OTU were decreased, and 3 were increased in response to avilamycin treatment (P = 0.005-0.047). Functional profiling of bacterial communities based on PICRUSt analysis revealed that 10 functional categories were enriched by avilamycin treatments, and 4 functional categories were decreased. In conclusion, our results demonstrated that the influence of avilamycin supplementation on the diversity, taxonomic composition, and functional profiles of the microbiota was evidently different in the ileum and cecum. These results further our understanding of the impact of AGP on the composition and activity of commensal bacteria in the chicken gastrointestinal tract to develop novel feeding strategies for improving animal health and performance.