Purpose: Empirical antibiotic therapy should follow the local bacterial susceptibility, and the breakpoints revisions of the antimicrobial susceptibility testing can reflect the changes in the antimicrobial susceptibility of bacteria. This study aimed to analyze whether the changes in the antimicrobial susceptibility to antibiotics caused by the breakpoint revision will affect the empirical antibiotic therapy and its appropriateness. Patients and Methods: A retrospective study was conducted among 831 hospitalized patients infected by Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa from April 10, 2018, to April 11, 2020. We evaluated the appropriateness of empirical therapy based on the antimicrobial susceptibility testing results. The rate of empirical use and appropriateness of fluoroquinolones was calculated, and logistic regression was used to analyze influencing factors of empirical use of fluoroquinolones. Results: The susceptibility rate of the three bacteria to levofloxacin (50.78% vs 32.06%) and ciprofloxacin (48.45% vs 21.90%) was decreased (P<0.001), while the resistance rate to levofloxacin (45.74% vs 58.73%) and ciprofloxacin (46.90% vs 66.67%) was increased (P<0.001) after the breakpoints revision. The empirical usage rate of fluoroquinolones in patients infected with Escherichia coli, Klebsiella pneumoniae, or Pseudomonas aeruginosa was 20.94%, which was influenced by the breakpoint revision (P=0.022), age (P=0.007), and the department (P=0.006); the appropriateness rate was 28.74%, affected by the pathogenic bacteria (P=0.001) and multidrug-resistant microorganism (P=0.001), department (P=0.024), and the length of stay before the empirical therapy (P=0.016). Conclusion: The susceptibility of bacteria to antibiotics has changed significantly after the breakpoint revision while the clinicians' empirical therapy failure to change accordingly, which results in the decrease of the appropriateness of empirical use. It is enlightened that we should conduct more research to evaluate the rational use of antibiotics from the laboratory perspective and carry out interventions such as education and supervision to strengthen the collaboration between the microbiology laboratories and clinicians to improve the empirical antibiotic therapy and slow down the antimicrobial resistance.