Teixobactin is a new antimicrobial of considerable importance in our fight against multidrug-resistant bacterial pathogens. No resistance to teixobactin has been reported, but we have demonstrated that Enterococcus faecalis exhibits remarkable tolerance to cell killing by teixobactin. E. faecalis is a leading cause of hospital-acquired infection and on the World Health Organization list of organisms for which new treatments are urgently required. Antimicrobial tolerance has been shown to facilitate the development of bona fide resistance, but the molecular mechanisms underpinning tolerance remain largely unknown. We have recently identified CroRS, a two-component regulatory system, as a key signalling pathway responding to teixobactin-induced stress. Strikingly, deletion of croRS completely abolished teixobactin tolerance. These data demonstrate the crucial role of CroRS in controlling the molecular response to teixobactin. The aim of this study is to determine the function of CroRS in antimicrobial tolerance and to develop CroRS as a new candidate for drug development.