A novelin vitrogut model was developed to better understand the in-teractions betweenEscherichia coliand the mouse cecal mucus commensal microbi-ota. The gut model is simple and inexpensive while providing an environment thatlargely replicates the nonadherent mucus layer of the mouse cecum. 16S rRNA geneprofiling of the cecal microbial communities of streptomycin-treated mice colonizedwithE. coliMG1655 orE. coliNissle 1917 and the gut model confirmed that the gutmodel properly reflected the community structure of the mouse intestine. Further-more, the results from thein vitrogut model mimic the results of publishedin vivocompetitive colonization experiments. The gut model is initiated by the colonizationof streptomycin-treated mice, and then the community is serially transferred in mi-crocentrifuge tubes in an anaerobic environment generated in anaerobe jars. Thenutritional makeup of the cecum is simulated in the gut model by using a mediumconsisting of porcine mucin, mouse cecal mucus, HEPES-Hanks buffer (pH 7.2), Cle-land’s reagent, and agarose. Agarose was found to be essential for maintaining thestability of the microbial community in the gut model. The outcome of competitionsbetweenE. colistrains in thein vitrogut model is readily explained by the “restau-rant hypothesis” of intestinal colonization. This simple model system potentially canbe used to more fully understand how different members of the microbiota interactphysically and metabolically during the colonization of the intestinal mucus layer.