The spatiotemporal structure of the human microbiome, proteome, and metabolome reflects and determines regional intestinal physiology and may have implications for disease. Yet, we know little about the distribution of microbes, their environment, and their biochemical activity in the gut because of reliance on stool samples and limited access to only some regions of the gut using endoscopy in fasting or sedated individuals. To address these deficiencies, we developed and evaluated a safe, ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion and maintains the viability of microbes from these locations. The collection of 240 intestinal samples from 15 healthy individuals using the device and subsequent multi-omics analyses revealed significant differences between microbes, phages, host proteins, and metabolites present in the intestines versus stool. Certain microbial taxa and gene classes were differentially enriched, and prophage induction was more prevalent in the intestines than in stool. The host proteome and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundancepredicted species that altered the bile acid pool through deconjugation. Furthermore,microbially conjugated bile acids displayed amino acid-dependent trends in concentration that were not apparent in stool. Overall, non-invasive longitudinal profilingof microbes, proteins, and bile acids along the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in humanphysiology and disease.