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BACKGROUND AND OBJECTIVES: Antibiotic use for suspected infection without a source occurs frequently in neonatal intensive care units (NICUs). Prolonged antibiotic use is associated with increased risk for late-onset sepsis, death, and emergence of multidrug-resistant bacteria. Our aim was to reduce antibiotic use for culture-negative sepsis (CNS) through interventions designed to decrease the number and length of antibiotic courses for CNS. METHODS: Our team used the Model for Improvement with sequential Plan-Do-Study-Act cycles to implement interventions in our level IV NICU. These included the creation of Antibiotic Guidelines, case audits, weekly antibiotic stewardship rounds, and biweekly review of CNS cases at staff meetings. All patients who received any dose of antibiotic and who had no major congenital anomalies were included. The primary outcome measures were CNS courses and antibiotic days of therapy (DOTs) per 1000 patient days (PDs). We tracked the number of antibiotic initiation events, re-initiation events, and antibiotic spectrum index as balancing measures. RESULTS: Antibiotic DOTs for CNS decreased significantly (81%) compared with baseline after study interventions. Subgroup analysis revealed this change was driven by a decrease in early-onset sepsis DOTs, with a reduction from 18.3 to 3.9 DOTs/1000 PDs. Overall antibiotic DOTs for the unit decreased from 232.5 to 176.7 DOTs/1000 PDs. There was no change in any of the prespecified balancing measures. CONCLUSIONS: This quality-improvement initiative, which prioritized case review and stewardship rounds to promote guideline adherence and reduce the treatment of CNS, resulted in a decrease in antibiotic use in a level IV NICU. [ABSTRACT FROM AUTHOR]