• Cohort 1: EVs miR-1246 expression can significantly discriminate between HC and PCa (AUC = 0.926), compared with serum PSA with AUC = 0.869.• Cohort 1 + 2: EVs miR-1246 observed a significant inverse correlation with PCa pathologic stage, specifically upregulated in aggressive prostate cancer.• miR-1246 was downregulated in PCa clinical tissues and cell lines and was selectively released into EVs.• Overexpression of miR-1246 in a PCa cell line significantly inhibited xenograft tumor growth in vivo and increased apoptosis and decreased proliferation, invasiveness, and migration in vitro. This evidence concerns the gene KLK3 and Familial prostate cancer.