Background: We aimed to explore the utility of prostate specific antigen (PSA) isoform [-2]proPSA and its derivatives for prediction of pathological outcome after radical prostatectomy (RP).Methods: Preoperative blood samples were prospectively and consecutively analyzed from 472 patients treated with RP for clinically localized prostate cancer at four medical centers. Measured parameters were PSA, free PSA (fPSA), fPSA/PSA ratio, [-2]proPSA (p2PSA), p2PSA/fPSA ratio and Prostate Health Index (PHI) (p2PSA/fPSA)*√PSA]. Logistic regression models were fitted to determine the accuracy of markers for prediction of pathological Gleason score (GS) ≥7, Gleason score upgrading, extracapsular extension of the tumor (pT3) and the presence of positive surgical margin (PSM). Results: Of 472 patients undergoing RP, 339 (72%) were found to have pathologic GS ≥ 7, out of them 178 (53%) experienced an upgrade from their preoperative GS=6. The findings of pT3 and PSM were present in 132 (28%) and 133 (28%) cases, respectively. At univariable analysis of all the preoperative parameters, PHI was the most accurate predictor of pathological GS ≥7, GS upgrading, pT3 disease and the presence of PSM. Adding of PHI into the base multivariable model increased significantly the accuracy for prediction of pathological GS and GS upgrading by 4.4% (p=0.015) and 5.0% (p=0.025), respectively. Conclusion: We found that PHI provides the highest accuracy in predicting prostate cancer aggressiveness and expansion of the tumor detected at final pathology. The ability of PHI to predict the risk of Gleason score upgrade may help to identify potentially high-risk patients among men with biopsy proven insignificant prostate cancer.