is a major nosocomial pathogen that can cause severe, toxin-mediated diarrhea and pseudomembranous colitis. Recent work has shown that exhibits heterogeneity in swimming motility and toxin production through phase variation by site-specific DNA recombination. The recombinase RecV reversibly inverts the flagellar switch sequence upstream of the operon, leading to the ON/OFF expression of flagellum and toxin genes. How this phenomenon impacts virulence remains unknown. We identified mutations in the right inverted repeat that reduced or prevented flagellar switch inversion by RecV. We introduced these mutations into R20291 to create strains with the flagellar switch "locked" in either the ON or OFF orientation. These mutants exhibited a loss of flagellum and toxin phase variation during growth , yielding precisely modified mutants suitable for assessing virulence . In a hamster model of acute infection, the phase-locked ON mutant caused greater toxin accumulation than the phase-locked OFF mutant but did not differ significantly in the ability to cause acute disease symptoms. In contrast, in a mouse model, preventing flagellum and toxin phase variation affected the ability of to colonize the intestinal tract and to elicit weight loss, which is attributable to differences in toxin production during infection. These results show that the ability of to phase vary flagella and toxins influences colonization and disease development and suggest that the phenotypic variants generated by flagellar switch inversion have distinct capacities for causing disease.