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Objective: To evaluate the change in consumption of specific antibiotics in a neonatal intensive care unit after the implementation of an antimicrobial stewardship program (ASP). Design: Retrospective cohort study between January 1, 2010, and December 31,2019. Setting: The neonatal intensive care unit at British Columbia Women’s Hospital (Vancouver Canada), a tertiary-care center. Patients: Admitted neonates prescribed antibiotics. Methods: We implemented an ASP with an early implementation phase starting in January 2014 (period 2) and a later phase starting in January 2017 (period 3). Patient demographics were collected, including birth weight, gestational age, history of necrotizing enterocolitis (NEC), and surgical operations from existing databases. Interrupted time-series analysis was used, and comparison of antibiotic days of therapy (DOT) averages were conducted across the preimplementation period (period 1), period 2, and period 3 regarding total patients and subgroups. Results: We identified 4,512 infants. There was a significant decrease in DOT from 472 (95% confidence interval [CI], 431–517) in period 1 to 405 (95% CI, 367–446) in period 2 to 313 (95% CI, 280–350) in period 3. We detected a significant decrease in the use of ampicillin, aminoglycosides, cloxacillin, and linezolid but not in vancomycin or cefotaxime. Subgroup analyses of infants Conclusions: The implementation of an ASP was associated with a significant decrease in the overall DOT and use of certain antibiotics. This study presents important targets for ongoing ASP work.