Background and Aims: Premature infants are at high risk for acute kidney injury (AKI) from various causes. The serum creatinine level has limitations in evaluating the renal function of premature infants. The purpose of this study is to evaluate whether urine biomarkers can be used to monitor development of AKI in premature infants. Methods: A prospective cohort study was conducted in premature infants born less than gestational age (GA) 37 weeks and admitted to the neonatal intensive care units of Kangnam Sacred Heart Hospital and Inha University Hospital. Urine biomarkers and serum creatinine were measured on postnatal day 1, 3, 5, 7, 10, and 14.. Urine biomarkers include neutrophil-gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), interleukin-8 (IL-8), liver fatty acid binding protein (L-FABP), cystatin-C (CysC), osteopontin (OPN), and epidermal growth factor (EGF). AKI was classified using modified Acute Kidney Injury Network (AKIN) definition. Results: A total of 83 infants were recruited. (male:female 1.4:1) Mean GA and birth weight was 30.5 ± 3.0 weeks and 1514 ± 549 g, respectively. AKIs occurred in 17 (20.5%) infants at mean age of 7.4 ± 2.6 days. When classified using the modified AKIN definition, 9 (12.0%) infants were classified as the stage 1, 5 (6.0%) and 2 (2.4%) infants were classified as the stage 2 and 3, respectively. Because AKI did not occur in infants born after GA 32 weeks, we compared demographic factors and urine biomarker between AKI and non-AKI groups under GA 32 weeks. There are no differences in baseline characteristics except GA in multivariate logistic regression analysis. Urine levels of NGAL, CysC, KIM-1, L-FABP and OPN were higher in AKI group than in non-AKI group before the onset of AKI. Urine levels of IL-8 were higher in AKI group than in non-AKI group at around the onset of AKI. Conversely, urine levels of EGF were lower in AKI group than in non-AKI group before the onset of AKI. Conclusion: In this study on premature infants, though there was no demographic risk factor for AKI except GA, several urine biomarkers were significantly different between AKI and non-AKI groups. Urine biomarkers could be useful to monitor renal function and predict AKI development in premature infants.