A common side effect of antibiotic treatment is the depletion of the patient’s endogenous gut microbiota, which provides critical protection from antibiotic-resistant pathogens that commonly contaminate hospitals, such as vancomycin-resistant Enterococcus (VRE). Understanding mechanisms of colonization resistance to VRE are therefore critical for the development of therapeutic interventions for patients undergoing antibiotic therapy. In this work, we took 2 different approaches to investigate how colonization resistance is formed by the commensal microbiota to protect the host. In the first approach, we utilized a recently designed tail cup device that functioned to prevent coprophagia during mouse experiments, enabling us to study the dynamics of VRE colonization without the influence of pathogen recycling. We found that VRE was capable of engrafting into the small intestine (SI) of antibiotic-treated mice even when coprophagia was prevented. Using a green-fluorescent protein expressing strain of Enterococcus faecium, we visualized the association of E. faecium in the SI of non-coprophagic mice and found small clusters of bacteria colonizing the epithelium directly. Application of these tail cups also allowed us to characterize how the large intestinal microbiota influences the immune system in the SI, increasing the stimulation of Reg3g production in the ileum and skewing the helper T cell populations of the mesenteric lymph nodes towards increased Th17 cells. Despite this, mice in tail cups were able to resolve VRE infection earlier than mice in mock cups, suggesting that the less diverse microbiota of their SI and subsequent decreased immune stimulation was still sufficient to confer protection from VRE when pathogen recycling is inhibited. Conversely, the increased stimulation and bacteria present in the coprophagic mice was less efficient at clearing VRE from the GI tract, likely due to the extremely high loads of VRE consumed during the course of infection. These studies indicate that VRE is quite sensitive to the resistance conferred by the SI immune system and microbiota, and that de-colonization of this area precedes clearance of the large intestine. In our second approach, we investigated ampicillin-resistance in antibiotic-naïve gut microbiota. By using commercially available mouse colonies, we were able to assess the presence of resistance to antibiotic treatment in healthy microbiota with no history of infection or antibiotic usage. Of the 7 different colonies we tested, we found that 1 was resistant to ampicillin-induced dysbiosis, enabling resistance to subsequent infection with VRE. We isolated 3 bacteria from the resistant microbiota, and demonstrated that they are sufficient to confer protection to an antibiotic-sensitive microbiota. These findings demonstrate that ampicillin-resistance can be found in diverse, complex microbiota derived from healthy populations unexposed to antibiotics. Altogether, this project brings insights into mechanisms of indirect resistance to colonization by VRE, showing that the SI is a distinct niche for colonization, and demonstrating the potential therapeutic role for antibiotic-resistant commensals.