IMPORTANCE Acute kidney injury (AKI) occurs commonly in preterm neonates and is associated with increased morbidity and mortality. OBJECTIVES To examine the association between caffeine citrate administration and AKI in preterm neonates in the first 7 days after birth and to test the hypothesis that caffeine administration would be associated with reduced incidence and severity of AKI. DESIGN, SETTING, AND PARTICIPANTS This studywas a secondary analysis of the Assessment ofWorldwide Acute Kidney Injury Epidemiology in Neonates (AWAKEN) study, a retrospective observational cohort that enrolled neonates born from January 1 to March 31, 2014. The dates of analysis were October 2016 to December 2017. The setting was an international, multicenter cohort study of neonates admitted to 24 participating level III or IV neonatal intensive care units. Participants met the original inclusion and exclusion criteria of the AWAKEN study. Additional exclusion criteria for this study included participants greater than or equal to 33 weeks' gestation at birth, admission after age 7 days, use of theophylline in the neonatal intensive care unit, or lack of data to define AKI. There were 675 preterm neonates available for analysis. EXPOSURE Administration of caffeine in the first 7 days after birth. MAIN OUTCOMES AND MEASURES The primary outcomewas the incidence of AKI (based on the modified neonatal Kidney Disease: Improving Global Outcomes [KDIGO] definition) in the first 7 days after birth. The hypothesis that caffeine administration would be associated with reduced AKI incidence was formulated before data analysis. RESULTS The study cohort (n = 675) was 55.4%(n = 374) male, with a mean (SD) gestational age of 28.9 (2.8) weeks and a mean (SD) birth weight of 1285 (477) g. Acute kidney injury occurred in 122 neonates (18.1%) in the first 7 days after birth. Acute kidney injury occurred less frequently among neonates who received caffeine than among those who did not (50 of 447 [11.2%] vs 72 of 228 [31.6%], P < .01). After multivariable adjustment, administration of caffeine remained associated with reduced odds of developing AKI (adjusted odds ratio, 0.20; 95%CI, 0.11-0.34), indicating that for every 4.3 neonates exposed to caffeine one case of AKI was prevented. Among neonates with early AKI, those receiving caffeine were less likely to develop stage 2 or 3 AKI (adjusted odds ratio, 0.20; 95%CI, 0.12-0.34). CONCLUSIONS AND RELEVANCE Caffeine administration in preterm neonates is associated with reduced incidence and severity of AKI. Further studies should focus on the timing and dosage of caffeine to optimize the prevention of AKI.