Daptomycin (DAP) is a novel lipopeptide antibiotic which exhibits excellent antibacterial activity against most clinically relevant Gram-positive bacteria, but the DAP-targeting protein molecules against host bacterial infection are far from clear. In order to discover bacterial protein response to DAP treatment, an iTRAQ-based quantitative proteomic analysis was applied to identify differential bacterial proteome profiling of Staphylococcus aureus (S. aureus) ATCC 25923 to 0.125μg/ml DAP exposure. Totally 51 bacterial proteins were significantly changed with DAP treatment, among which 34 proteins were obviously up-regulated and 17 proteins were down-regulated. Meanwhile, 139 bacterial cell membrane (CM) proteins were identified, and 7 CM proteins were significantly altered to decrease CM potential to disrupt bacterial cell membrane. Especially the up-regulation of NDK and down-regulation of NT5 in several S. aureus strains are validated to be a universal variation tendency response to DAP treatment. Under DAP exposure, bacterial membrane potential is decreased and cell membrane is disrupted, and bacterial chromosome is aggregated, which contributes to bacterial DNA rapid release and induces bacteria death within 2-5h. In general, multiple bacterial protein expressions are changed in response to DAP antibiotic exposure, which disrupts host bacterial physiology by multiple cellular levels. To our knowledge, this is the first time to exactly identify infectious bacterial proteins in response to DAP antibiotic action. Our findings help better understand DAP antibacterial mechanism and develop novel DAP derivatives against the upcoming antibiotic-resistant bacterial infection. DAP is a novel lipopeptide antibiotic that it exhibits excellent in vitro activity against most clinically relevant Gram-positive bacteria, and the investigations on its pharmaceutical action mode of DAP have dramatically increased in the past decade due to its unique antimicrobial mechanism. However, the target molecules of DAP acting on the infectious bacteria, are far from clear. The state-of-the-art quantitative proteomic technologies provide new avenues to uncover underlying mechanism of antibiotics. Our research main aims to identify bacterial proteome profiling of host strain S. aureus response to DAP treatment through an iTRAQ-based quantitative proteomic analysis, which contributes to understand DAP efficient antibacterial activity and the microbial-antibiotic interactions. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.