• 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. Here, KLK3 is linked to posterior cortical atrophy.