A crucial step in antimicrobial development pipelines is proof of functionality in the host environment. Antibiotics targeting fatty acid synthesis (FASII) of the major pathogen Staphylococcus aureus actively inhibit FASII but do not prevent in vivo growth, as bacteria compensate the FASII block by using environmental fatty acids. We report that S. aureus responds to FASII antibiotic treatment by major shifts in expression that correlate with improved fitness but decreased virulence, and with altered bacterial killing in an insect infection model. Proteomic kinetics and phosphoproteomic analyses show that anti-FASII provokes massive protein reprogramming compared to non-treated S. aureus , while growth is robust in both conditions. Anti-FASII adaptation leads to overall increases in stress response functions and provides greater resistance to peroxide, as produced during host infection. Moreover, S. aureus adaptation to anti-FASII is accelerated by pre-treatment with peroxides. In contrast, virulence factor levels are decreased. In keeping with the observed phenotypes, anti-FASII-adapted S. aureus are slower to kill their host in a Galleria mellonella infection model than non-treated bacteria, but are not eliminated. Thus, anti-FASII adapted cells might be better prepared for survival and less equipped to damage the host. If not eliminated, anti-FASII reprogrammed S. aureus populations might provide a bacterial reservoir for the emergence of chronic infections. Author Summary Bacterial resistance or adaptation to currently used antibiotics is a main reason for treatment failure. Our work on the major human pathogen Staphylococcus aureus revealed that antibiotics targeting fatty acid synthesis (FASII) effectively reach their targets, but do not stop growth. Here we analyzed the expression and infection capacities of S. aureus populations that escape anti-FASII inhibition by adapting to the antibiotic. Remarkably, anti-FASII-adapted bacteria show massive expression changes, but grow normally. Once adapted, bacteria produce greater amounts of stress response proteins, while virulence factor levels or activities are lower. Accordingly, anti-FASII-adapted S. aureus provoke slower killing in an insect infection model, but bacteria continue to multiply. If not eliminated, anti-FASII reprogrammed S. aureus populations could provide a bacterial reservoir for establishment of chronic infections.