In the current study, the gls24 disruption mutant TX10100, previously shown to be more sensitive to bile salts and attenuated in a mouse peritonitis model, showed an approximately fivefold higher 50% infective dose than wild-type OG1RF in a rat endocarditis model. When administered as a mixture, TX10100, unlike a downstream glsB mutant, was significantly outnumbered by OG1RF in vegetations, organs, and blood, despite being inoculated in greater numbers. These results indicate that gls24 is important in the pathogenesis of enterococcal endocarditis.