The purpose of the present study was to establish the development of acute kidney injury (AKI) and evaluate the usefulness of kidney-specific biomarkers in diagnosing AKI in premature calves with respiratory distress syndrome (RDS). Ten-term healthy and 70 premature calves with RDS were enrolled. Clinical examination, blood gases, and chemical analysis were performed at admission and 72 h. Serum concentrations of blood urea nitrogen (BUN), creatinine (Cre), phosphorus (P), cystatin-C (Cys-C), neutrophil gelatinase-associated lipocalin (NGAL), uromodulin (UMOD), and liver-type fatty acid-binding protein (L-FABP) were measured to evaluate kidney injury. Our findings showed that 38.5% of the premature calves with RDS developed AKI. The RDS-AKI group had a 4-fold higher mortality risk than the RDS-non-AKI group. Cys-C, with 90% and 89% specificity, and NGAL, with 100% sensitivity and 85% specificity, were the most reliable biomarkers to determine AKI in premature calves. The usefulness of any biomarker to predict mortality was not found to be convincing. In conclusion, AKI can develop as a consequence of hypoxia in premature calves and may increase the risk of mortality. In addition, serum Cys-C and NGAL concentrations may be useful in the diagnosis of AKI in premature calves with RDS.