BACKGROUND: MicroRNAs (miRNAs) play a key role in regulating gene expression in Crohn's disease (CD). Although several studies have identified miRNAs with biomarker potential, an exhaustive characterization of the miRNAome in CD is still lacking. We performed the largest miRNA profiling effort to date to analyze miRNA variability across intestinal tissues, disease activity status, and infliximab treatment in CD. METHODS: We generated 119 transcriptomic profiles from the terminal ileum and left colon biopsies of 30 individuals (10 with active CD, 10 with quiescent CD, and 10 healthy controls). Half of the samples were cultured ex vivo with infliximab, and the remaining half with basal medium. Using variance analyses and linear mixed differential expression models, we explored the determinants of miRNAome variability in CD. We also generated infliximab response signatures to identify candidates and examine interactions between miRNAs and the coding transcriptome. RESULTS: Tissue location, and patient-specific effects, were the main factors in miRNA variability in CD, with some differentially expressed miRNAs involved in epithelial-mesenchymal transition (miR-200s, miR-429). We identified 9 miRNAs with treatment-responsive behaviour, particularly to the terminal ileum of active CD cases. Although the changes observed in active CD cases suggest that many alterations are not offset by infliximab incubation, we described 13 miRNAs-mRNA pairs with potential involvement in the anti-tumor necrosis factor (TNF) treatment, 7 of which have been already validated. CONCLUSIONS: A comprehensive miRNA profiling revealed significant intestinal tissue-specific variability and identified key alterations in the miRNA-mRNA interactome that might be involved in therapeutic response to anti-TNF in CD.