Enterococcus faecalis and E. faecium are gram-positive human pathogens that can live in the gastrointestinal tract of food animals. Vancomycin-resistant enterococci are an increasing threat to humans as a nosocomial infection, as well as a reservoir of antibiotic resistance genes. Ionophores are feed-grade antimicrobials that are widely used to enhance the ruminal fermentation efficiency via inhibiting gram-positive bacteria by dissipating ion and proton gradients. Some bacteria can become resistant to ionophores, and this has prompted concerns about whether ionophore resistance can enhance antibiotic resistance in intestinal bacteria. Since enterococci are normal members of the ruminant intestinal tract and function as an antibiotic resistance reservoir, the present study investigated whether treatment with the most commonly used ionophores affected the growth of enterococci, and whether ionophore-resistant enterococci developed. Ionophores do inhibit the growth of enterococci in pure culture, but in our study did not alter populations in mixed ruminal bacterial culture. Ionophore-resistant isolates were not isolated during this study from pure or mixed cultures. Our results indicate that the role of ionophores in the dissemination of antibiotic resistance genes through the intestinal Enterococcus spp. appears to be limited.