Current techniques for studying gut microbiota are unable to answer some important microbiology questions, like how different bacteria grow and divide in the gut. Here we propose a novel method, which integrates the use of sequential D-amino acid-based in vivo metabolic labeling and fluorescence in situ hybridization (FISH), for unveiling the growth and division patterns of gut bacteria. After sequential gavage of two D-amino acid-based probes containing different fluorophores to mice, the dually labeled peptidoglycan of gut bacteria provides temporal information of their cell wall synthesis. Following taxonomic identification with FISH probes, the growth and division patterns of the corresponding bacterial taxa, including species that cannot be cultured separately in vitro, are revealed. Our method offers a facile yet powerful tool for investigating the indigenous growth of gut microbiota, which will help expanding our scope of bacterial cytology and deciphering some basic microbiology of these gut “dark matters”.