Antibiotics with novel bactericidal mechanisms of action are urgently needed. The antibiotic acyldepsipeptide 4 (ADEP4) activates the ClpP protease and causes cells to self-digest. The effects of ADEP4 and ClpP activation have not been characterized sufficiently for the enterococci， which are important pathogens known for high levels of acquired and intrinsic antibiotic resistance. In the present study， ADEP4 was found to be potently active against both and ， with MICs of 0.016 μg/ml and 0.031 μg/ml， respectively. ClpP purified from was found to bind ADEP4 in a surface plasmon resonance analysis， and ClpP activation by ADEP4 was demonstrated biochemically with a β-casein digestion assay. In addition， ClpP was crystallized in the presence of ADEP4， revealing ADEP4 binding to ClpP in the activated state. These results confirm that the anti-enterococcal activity of ADEP4 occurs through ClpP activation. In killing curve assays， ADEP4 was found to be bactericidal against stationary-phase vancomycin-resistant (VRE) strain V583， and resistance development was prevented when ADEP4 was combined with multiple classes of approved antibiotics. ADEP4 in combination with partnering antibiotics also eradicated mature VRE biofilms within 72 h of treatment. Biofilm killing with ADEP4 antibiotic combinations was superior to that with the clinically used combinations ampicillin-gentamicin and ampicillin-daptomycin. In a murine peritoneal septicemia model， ADEP4 alone was as effective as ampicillin. ADEP4 coadministered with ampicillin was significantly more effective than either drug alone. These data suggest that ClpP-activating antibiotics may be useful for treating enterococcal infections.