Abstract The continuously remodeled extracellular matrix (ECM) plays a pivotal role in gastrointestinal health and disease, yet its precise functions remain elusive. In this study, we employed laser capture microdissection combined with low-input proteomics to investigate ECM remodeling during Salmonella -driven inflammation. To complement this, we probed how fibronectin fiber tension is altered using a mechanosensitive peptide probe. While fibronectin fibers in healthy intestinal tissue are typically stretched, many lose their tension in intestinal smooth muscles only hours after infection, despite the absence of bacteria in that area. In contrast, within the mucosa, where Salmonella is present starting 12 h post infection, fibronectin fiber relaxation occurred exclusively during late-stage infection at 72 h and was localized to already existing clusters of infiltrated neutrophils. Using N-terminomics, we identified three new cleavage sites in fibronectin in the inflamed cecum. The unique, tissue layer-specific changes in the molecular compositions and ECM fiber tension revealed herein might trigger new therapeutic strategies to fight acute intestinal inflammation.