<p>Metagenomic sequencing and metabolomics of fecal matter have revealed the impact of the gut microbiome on health and disease. In addition to microbiota, feces also contain shed or exfoliated host epithelial, secretory and immune cells, but RNA profiling of these cells is challenging owing to degradation and cross-contamination. Here we introduce exfoliome sequencing (Foli-seq) to profile fecal exfoliated eukaryotic messenger RNAs (feRNAs) originating from the upper and lower gastrointestinal regions and show that this ‘fecal exfoliome’ harbors stable RNAs that reflect intestinal and immune function. By selectively amplifying targeted transcripts, Foli-seq demonstrates robust, accurate, sensitive and quantitative measurement of feRNAs. In murine colitis models, feRNA reveals temporal processes of epithelial damage, immune response and intestinal recovery specific to different types of gut inflammation. Simultaneous exfoliome and microbiome profiling uncovers a dense host–microbe interaction network. Moreover, we demonstrate stratification of patients with inflammatory bowel disease into subgroups that correlate with disease severity. Fecal Foli-seq is a noninvasive strategy to longitudinally study the gut and profile its health.</p>