Acute kidney injury (AKI) is detrimental after cardiac surgery. In this multicenter study, the novel biomarker hemojuvelin (HJV) was evaluated for AKI prediction following cardiac surgery. Urinary HJV, neutrophil gelatinase-associated lipocalin (NGAL), and urinary creatinine were measured in 151 patients after surgery. The outcomes of advanced AKI (KDIGO stages 2 and 3) and all causes of in-hospital mortality as the composite outcome were recorded. Areas under the receiver operator characteristic curves (AUC) and a multivariate generalized additive model (GAM) were applied to predict these outcomes of interest. Urinary HJV differentiated patients with/without AKI, advanced AKI or composite outcome after surgery (p < 0.001, by a generalized estimating equation) in this study. At three hours post-surgery, urinary HJV predicted advanced AKI (p < 0.001) and composite outcome (p < 0.001) with corresponding AUC values of 0.768 and 0.828, respectively. The performance of creatinine-adjusted HJV was also superior to NGAL in predicting advanced AKI (AUC = 0.784 and 0.694; p = 0.037) and composite outcome (AUC = 0.842 and 0.676; p = 0.002). The integration of HJV into the Cleveland Clinic score for advanced AKI led to a significant increase in risk stratification (net reclassification improvement [NRI] = 0.598; p < 0.001).