A neonate born at 25 + 1/7 weeks developed ventilator-associated pneumonia at 29 + 3/7 weeks post-menstrual age with Escherichia coli that was originally sensitive to gentamicin. After 3 days of treatment with gentamicin, the minimum inhibitory concentration (MIC) changed from less than 1 mg/L to more than 16 mg/L. It appears that suboptimal gentamicin dosing led to the development of gentamicin resistance. As the patient was not improving clinically, the antibiotics were changed once the gentamicin resistance was identified. To minimize resistance and treatment failure, clinicians should consider the patient-specific pharmacokinetic parameters, achieved peak level, and the amount of time the gentamicin level will remain below the MIC of the organism being treated.