The intestinal epithelium is a highly structured tissue composed of repeating crypt-villus units. Enterocytes perform the diverse tasks of absorbing a wide range of nutrients while protecting the body from the harsh bacterium-rich environment. It is unknown whether these tasks are spatially zonated along the villus axis. Here, we extracted a large panel of landmark genes characterized by transcriptomics of laser capture microdissected villus segments and utilized it for single-cell spatial reconstruction, uncovering broad zonation of enterocyte function along the villus. We found that enterocytes at villus bottoms express an anti-bacterial gene program in a microbiome-dependent manner. They next shift to sequential expression of carbohydrates, peptides, and fat absorption machineries in distinct villus compartments. Finally, they induce a Cd73 immune-modulatory program at the villus tips. Our approach can be used to uncover zonation patterns in other organs when prior knowledge of landmark genes is lacking. Display omitted •Laser capture microdissection reveals a large panel of enterocyte landmark genes•These genes are used to spatially localize single RNA-sequenced enterocytes•Enterocyte function is broadly zonated along the villus axis•Enterocytes traverse a series of cell states during their migration along the villus A broadly applicable single-cell spatial transcriptomics approach reveals broad regional and functional heterogeneity of small intestinal enterocytes.