Dexmedetomidine is being increasingly used as a primary or adjunctive sedative agent in neonates. There are a paucity of high-quality, high-resolution physiologic data during administration, despite significant potential cardiorespiratory effects. Term and preterm infants admitted between January 2018 and July 2020 were screened for dexmedetomidine exposure. Prospectively recorded vital signs (heart rate, oxygenation, arterial blood pressure) were cross-matched with pharmacy records to identify infants with data available 24 h before and 48 h after drug initiation. Vital sign data were processed via a standardized pipeline to (a) remove missing data, (b) obtain baseline averages of vital signs for 24 h preceding dexmedetomidine, and (c) calculate the hourly mean deviation from the baseline for the 48 h following initiation of dexmedetomidine. Infants were clustered by postmenstrual age (preterm ≤ 35 weeks; term > 35 weeks). 72 infants were identified with mean gestational age of 32 weeks and mean ± SD birth weight of 1976 ± 1341 g. Although both groups of infants experienced bradycardia, heart rate in term infants dropped faster and reached a nadir 5 beats per minute lower, before converging at a common deviation of − 10 beats per minute. No hypo- or hypertension was noted in either group. Unexpected instability of oxygenation occurred in a subset of preterm infants, requiring escalation of respiratory support. Administration of dexmedetomidine results in differential timing and magnitude of bradycardia in term and preterm infants, no major impact on blood pressure, and a surprising instability of oxygenation in preterm infants, requiring increased ventilatory support. Further investigation is warranted.