Although the anterior nares are the most common site of staphylococcal carriage, persistent colonization of Staphylococcus aureus in the GI tract and other body sites contributes to the limited success of topical therapies for eradication of carriage. Furthermore, the GI tract or rectum may be a reservoir for USA300 S. aureus strains. We developed a murine model of S. aureus GI colonization to identify bacterial factors that promote GI colonization. Mice given intranasal inocula ranging from 10⁹ to 10⁵ CFU S. aureus Newman colonized the mouse GI tract for 3 weeks. Isogenic mutants lacking capsular polysaccharide (Dcap5O) or wall anchored proteins (srtA::ermB) showed a ~0.5 log reduction in GI colonization compared to strain Newman. In contrast, a mutant lacking wall teichoic acid (tagO::ermB) showed a 1.7 log reduction in GI colonization compared to strain Newman. Similarly, a tagO mutant of S. aureus SA113 showed a 1.4 log reduction in GI colonization compared to the parental strain. Thus, wall teichoic acid plays an important role in both nasal and GI colonization of S. aureus. To determine whether the ACME element unique to USA300 strains promotes GI colonization, we constructed an isogenic mutant of a USA300 isolate with SCCmec and ACME deleted. In separate mouse groups, the parent and mutant colonized the GI tract in similar numbers; nasal colonization was poor for both strains. However, the results of competition experiments indicated that the deletion of both SCCmec and ACME significantly reduced the fitness of USA300 strains to colonize the murine GI tract.