Abstract: BACKGROUND: To date, there has been limited work evaluating the total cumulative effective radiation dose received by infants in the neonatal intensive care unit. Most previous publications report that the total radiation dose received falls within the safe limits but does not include all types of ionizing radiation studies typically performed on this vulnerable patient population. We aimed to provide an estimate of the cumulative effective ionizing radiation dose (cED) in microSieverts (μSv) received by premature infants ≤32 weeks from diagnostic studies performed throughout their NICU stay, and predictors of exposures. METHODS: Retrospective chart review from 2004–2011. Data included demographics, gestational age (GA), birth weight (BW), length of stay (LOS), clinical diagnosis, and radiological studies. RESULTS: 1045 charts were reviewed. Median GA = 30.0 weeks (SD 2.7, range 22.0–32.6). Median BW = 1340.0 grams (SD 445.4, range 420–2470). Median number of radiographic studies = 9 (SD 28.5, range 0–210). Median cED = 162μSv (range 0–9248). The cED was positively associated with LOS (p < 0.001) and inversely correlated with GA (p < 0.001) and BW (p < 0.001). Infants with intestinal perforation had the highest median cED 1661μSv compared to 162μSv for others (p < 0.001). CONCLUSION: Our results provide an estimate of the cumulative effective radiation dose received by premature infants in a level 4 neonatal intensive care unit from all radiological studies involving ionizing radiation and identifies risk factors and predictors of such exposure. Radiation exposure in NICU is highest among the most premature and among infants who suffer from intestinal perforation.