Abstract The infection of drug resistant bacteria seriously threats to public health, which makes it more urgent to find novel antibacterial compounds. Compared with traditional development approaches, drug repurposing provides a faster and more effective strategy to find new antimicrobial agents. Here, we screened an FDA-approved small-molecule library upon S. aureus, and identified crizotinib as an antimicrobial agent. We confirmed the antibacterial activities of crizotinib in vitro and in vivo by MIC, growth curve, SEM and mice experiment. At the same time, crizotinib also showed low tendency to develop resistance. Mechanistically, quantitative proteomics, bioinformatics analyses, qRT-PCR and flow cytometry biochemical validation experience confirmed that crizotinib exerted its antibacterial effects by interfering pyrimidine metabolism and disrupting DNA synthesis eventually. Furthermore, our data from drug affinity responsive target stability, bio-layer interferometry and a series of functional assays demonstrated that crizotinib could target PyrG directly in pyrimidine metabolism pathway. Taken together, our results indicated that crizotinib could be a potential antimicrobial agent to treat Gram-positive bacterial infections in the future.