The consequences on glycopeptide activity of low-level resistance to vancomycin due to VanE-type resistance were evaluated in vitro and in experimental endocarditis caused by Enterococcus faecalis BM4405 (MICs of vancomycin and teicoplanin: 16 and 0.5 µg/ml, respectively), its susceptible derivative BM4405-1, and susceptible E. faecalis JH2-2. After 24 h of incubation, vancomycin at 8 µg/ml was not active against E. faecalis BM4405 whereas it was bacteriostatic against strains BM4405-1 and JH2-2. Against all three strains, vancomycin at 30 µg/ml and teicoplanin at 8 or 30 µg/ml were bacteriostatic but bactericidal when combined with gentamicin. In rabbits with aortic endocarditis due to VanE-type resistant strain BM4405, treatment with a standard dose of vancomycin generated subinhibitory plasma concentrations (i.e., peak of 36.3 ± 2.1 µg/ml and trough of 6.0 ± 2.2 µg/ml) and led to no significant reduction in mean aortic valve vegetation counts compared to no treatment of control animals. In contrast, a higher dosing regimen of vancomycin (i.e., resulting in a peak of 38.3 ± 5.2 µg/ml and a trough of 15.0 ± 8.3 µg/ml), providing plasma concentrations above the MIC for the entire dosing interval, led to significant and similar activities against all three strains, which were enhanced by combination with gentamicin. Treatment with teicoplanin led to results similar to those obtained with vancomycin at a high dose. No subpopulations with increased resistance to glycopeptides were selected in vitro or in vivo. In conclusion, the use of a high dose of vancomycin was necessary for the treatment of experimental enterococcal endocarditis due to VanE-type strains.