The internal environment of the gallbladder has been considered extremely unfavorable for bacterial growth, and the microbial profile of the gallbladder still unknown. By high-throughput sequencing of the bacterial 16S rRNA gene, we studied the microbial profile of the gallbladder from healthy rabbits before and after weaning. Moreover, we investigated the difference of microbiota between the gallbladder and gut. Our results showed that the gallbladder was dominantly populated by Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria in the phylum throughout the developmental stages of rabbits. The adult rabbits showed higher species richness and exhibited higher bacterial diversity than rabbits before weaning based on the results of alpha diversity. Beta diversity analyses indicated differences in the bacterial community composition between different developmental stages. In the comparison of the gallbladder and feces, Firmicutes and Bacteroidetes were dominant in the phylum, as they were present in about 61% and 21% of the feces, respectively. Conversely, in the gallbladder, Firmicutes was the most dominant (about 41%), and Bacteroidetes and Proteobacteria were present in about 16% and 22% of the gallbladder, respectively. The Unweighted UniFrac Principal Coordinate Analysis results illustrated samples clustered into 2 categories: the gallbladder and feces. Our study might provide a foundation for knowledge on gallbladder microbiota for the first time and a basis for further studies on gallbladder and intestinal health.