© 2018 by The Society for Healthcare Epidemiology of America. All rights reserved. Objective Due to concerns over increasing fluoroquinolone (FQ) resistance among gram-negative organisms, our stewardship program implemented a preauthorization use policy. The goal of this study was to assess the relationship between hospital FQ use and antibiotic resistance.Design Retrospective cohort.Setting Large academic medical center.Methods We performed a retrospective analysis of FQ susceptibility of hospital isolates for 5 common gram-negative bacteria: Acinetobacter spp., Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Primary endpoint was the change of FQ susceptibility. A Poisson regression model was used to calculate the rate of change between the preintervention period (1998-2005) and the postimplementation period (2006-2016).Results Large rates of decline of FQ susceptibility began in 1998, particularly among P. aeruginosa, Acinetobacter spp., and E. cloacae. Our FQ restriction policy improved FQ use from 173 days of therapy (DOT) per 1,000 patient days to <60 DOT per 1,000 patient days. Fluoroquinolone susceptibility increased for Acinetobacter spp. (rate ratio [RR], 1.038; 95% confidence interval [CI], 1.005-1.072), E. cloacae (RR, 1.028; 95% CI, 1.013-1.044), and P. aeruginosa (RR, 1.013; 95% CI, 1.006-1.020). No significant change in susceptibility was detected for K. pneumoniae (RR, 1.002; 95% CI, 0.996-1.008), and the susceptibility for E. coli continued to decline, although the decline was not as steep (RR, 0.981; 95% CI, 0.975-0.987).Conclusions A stewardship-driven FQ restriction program stopped overall declining FQ susceptibility rates for all species except E. coli. For 3 species (ie, Acinetobacter spp, E. cloacae, and P. aeruginosa), susceptibility rates improved after implementation, and this improvement has been sustained over a 10-year period.