Pharmacokinetic (PK) data to guide cefazolin dosing in premature infants are virtually non-existent. Therefore, we aimed to characterize cefazolin PK in infants aged ≤32 weeks of gestation at birth.We conducted a prospective, open-label PK and safety study of cefazolin in infants ≤32 weeks gestation from a University Medical Center. We administered intravenous cefazolin and collected both timed and scavenged blood samples. We analyzed data using non-linear mixed effect modeling and simulated several dosage regimens to achieve target concentrations against methicillin-susceptible Staphylococcus aureus.We analyzed 40 samples from nine infants and observed that premature infants had lower clearance and greater volume of distribution for cefazolin compared to older children. The median (range) individual Bayesian estimates were 0.03 L/h/kg (0.01-0.08) for clearance and 0.39 L/kg (0.31-0.52) for volume.Simulations suggested reduced cefazolin dosing based on postmenstrual age achieve target concentrations and potentially reduce unnecessary exposure.