Due to the challenges to acquire detailed spatial information of gut bacteria in situ, microbiota’s three-dimensional (3D) distributions in the gut remain largely uncharted. Here, we propose a tissue clearing-based and D-amino acid labeling-facilitated (TiDaL) strategy that combines a novel microbiota in vivo labeling protocol, CUBIC-based tissue clearing and whole-mount tissue imaging, to achieve 3D imaging of indigenous gut microbiota. We demonstrate high resolution 3D acquisition of their biogeography in different gut sections, and present quantitative spatial details in relation to the host epithelium. We unexpectedly observe microbiota in the small intestine crypts which were thought to be bacteria-free. Moreover, significant bacterial overgrowth in the first two-thirds of the small intestine is detected in an enteritis model. We expect that this quantitative 3D imaging strategy for native gut microbiota will provide insightful information into the host-microbiota interactions.