The diarylquinoline TMC207 kills Mycobacterium tuberculosis by specifically inhibiting ATP synthase. We show here that human mitochondrial ATP synthase (50% inhibitory concentration [IC50] of >200 μM) displayed more than 20,000-fold lower sensitivity for TMC207 compared to that of mycobacterial ATP synthase (IC50 of 10 nM). Also, oxygen consumption in mouse liver and bovine heart mitochondria showed very low sensitivity for TMC207. These results suggest that TMC207 may not elicit ATP synthesis-related toxicity in mammalian cells. ATP synthase, although highly conserved between prokaryotes and eukaryotes, may still qualify as an attractive antibiotic target.