ABSTRACT Daptomycin is used off-label for enterococcal infections; however, dosing targets for resistance prevention remain undefined. Doses of 4 to 6 mg/kg of body weight/day approved for staphylococci are likely inadequate against enterococci due to reduced susceptibility. We modeled daptomycin regimens in vitro to determine the minimum exposure to prevent daptomycin resistance (Dap r ) in enterococci. Daptomycin simulations of 4 to 12 mg/kg/day (maximum concentration of drug in serum [ C max ] of 57.8, 93.9, 123.3, 141.1, and 183.7 mg/liter; half-life [ t 1/2 ] of 8 h) were tested against one Enterococcus faecium strain (S447) and one Enterococcus faecalis strain (S613) in a simulated endocardial vegetation pharmacokinetic/pharmacodynamic model over 14 days. Samples were plated on media containing 3× the MIC of daptomycin to detect Dap r . Mutations in genes encoding proteins associated with cell envelope homeostasis ( yycFG and liaFSR ) and phospholipid metabolism (cardiolipin synthase [ cls ] and cyclopropane fatty acid synthetase [ cfa ]) were investigated in Dap r derivatives. Dap r derivatives were assessed for changes in susceptibility, surface charge, membrane depolarization, cell wall thickness (CWT), and growth rate. Strains S447 and S613 developed Dap r after simulations of 4 to 8 mg/kg/day but not 10 to 12 mg/kg/day. MICs for Dap r strains ranged from 8 to 256 mg/liter. Some S613 derivatives developed mutations in liaF or cls . S447 derivatives lacked mutations in these genes. Dap r derivatives from both strains exhibited lowered growth rates, up to a 72% reduction in daptomycin-induced depolarization and up to 6-nm increases in CWT ( P < 0.01). Peak/MIC and AUC 0–24 /MIC ratios (AUC 0–24 is the area under the concentration-time curve from 0 to 24 h) associated with Dap r prevention were 72.1 and 780 for S447 and 144 and 1561 for S613, respectively. Daptomycin doses of 10 mg/kg/day may be required to prevent Dap r in serious enterococcal infections.