There is an urgent need to develop potent antimicrobials for the treatment of infections caused by antibiotic-resistant bacterial pathogens. Paenipeptin C' (C8-Pat) is a novel linear lipopeptide recently discovered by our group. The objectives of this study were to determine the time-kill kinetics of paenipeptin C' against Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 and to investigate its mechanism of action.,Paenipeptin C' was synthesized by solid-phase peptide synthesis and purified by HPLC to homogeneity. Paenipeptin C' showed concentration-dependent bactericidal activity against P. aeruginosa and S. aureus. Purified lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria and lipoteichoic acid (LTA) from Gram-positive bacteria significantly decreased the antibacterial activity of paenipeptin C', which indicated that LPS and LTA on cell surfaces are likely the initial binding targets of this antibiotic agent. Moreover, paenipeptin C' damaged bacterial cytoplasmic membranes, as evidenced by the depolarization of membrane potential and leakage of intracellular potassium ions. Specifically, paenipeptin C' at 32-64 μg/mL caused a significant membrane potential depolarization in P. aeruginosa and S. aureus. This antibiotic at 64-128 μg/mL rapidly induced the release of intracellular potassium ions from P. aeruginosa and S. aureus. Transmission electron microscopy imaging results showed that paenipeptin C' at bactericidal concentrations perturbed the cell envelopes, leading to the loss of intracellular contents.,Therefore, paenipeptin C' exerts its bactericidal effect through damaging bacterial cytoplasmic membrane.