AbstractImproved prostate cancer prognostic biomarkers are urgently needed. We previously identified the four-miRNA prognostic biomarker panel MiCaP ((miR-23a-3p × miR-10b-5p)/(miR-133a-3p × miR-374b-5p)) for prediction of biochemical recurrence (BCR) after radical prostatectomy (RP). Here, we identified an optimal numerical cut-off for MiCaP dichotomisation using a training cohort of 475 RP patients and tested this in an independent cohort of 281 RP patients (PCA281). Kaplan–Meier, uni- and multivariate Cox regression analyses were conducted for multiple endpoints: BCR, metastatic-(mPC) and castration-resistant prostate cancer (CRPC), prostate cancer-specific (PCSS) and overall survival (OS). Functional effects of the four MiCaP miRNAs were assessed by overexpression and inhibition experiments in prostate cancer cell lines. We found the numerical value 5.709 optimal for MiCaP dichotomisation. This was independently validated in PCA281, where a high MiCaP score significantly [and independent of the Cancer of the Prostate Risk Assessment Postsurgical (CAPRA-S) score] predicted BCR, progression to mPC and CRPC, and PCSS, but not OS. Harrell’s C-index increased upon addition of MiCaP to CAPRA-S for all endpoints. Inhibition of miR-23a-3p and miR-10b-5p, and overexpression of miR-133a-3p and miR-374b-5p significantly reduced cell survival. Our results may promote future implementation of a MiCaP-based test for improved prostate cancer risk stratification.