Extraintestinal pathogenic Escherichia coli (ExPEC) strains are typically benign within the  mammalian gut, but can disperse to extraintestinal sites to cause disease. As occupation of the  intestinal tract is often a prerequisite for ExPEC-mediated pathogenesis, we set out to understand  how ExPEC colonizes this niche. A screen using transposon sequencing (Tn-seq) was performed  to search for genes within the ExPEC isolate F11 that are important for growth in intestinal mucus, which is thought to be a major source of nutrients for E. coli in the gut. Multiple genes  that contribute to ExPEC fitness in mucus broth were identified, with genes that are directly or  indirectly associated with fatty acid beta-oxidation pathways being especially important. One of the identified mucus-specific fitness genes encodes the rhomboid protease GlpG. In vitro, we found that the disruption of glpG had polar effects on the downstream gene glpR, which encodes  a transcriptional repressor of factors that catalyze glycerol degradation. Mutation of either glpG or glpR impaired ExPEC growth in mucus and on plates containing the long chain fatty acid  oleate as the sole carbon source. In contrast, in a mouse gut colonization model in which the  natural microbiota is unperturbed, the disruption of glpG but not glpR significantly reduced  ExPEC survival. This work reveals a novel biological role for a rhomboid protease, and  highlights new avenues for defining mechanisms by which ExPEC colonize the mammalian  gastrointestinal tract.