Our recent determination of a microRNA (miRNA) expression signature in prostate cancer (PCa) revealed that miR-205-5p was significantly reduced in PCa tissues and that it acted as an antitumor miRNA. The aim of this study was to identify oncogenic genes and pathways in PCa cells that were regulated by antitumor miR-205-5p. Genome-wide gene expression analyses and in silico miRNA database searches showed that 37 genes were putative targets of miR-205-5p regulation. Among those genes, elevated expression levels of seven in particular (HMGB3, SPARC, MKI67, CENPF, CDK1, RHOU, and POLR2D) were associated with a shorter disease-free survival in a large number of patients in the The Cancer Genome Atlas (TCGA) database. We focused on high-mobility group box 3 (HMGB3) because it was the most downregulated by ectopic expression of miR-205-5p in PC3 cells and its expression was involved in PCa pathogenesis. Luciferase reporter assays showed that HMGB3 was directly regulated by miR-205-5p in PCa cells. Knockdown studies using si-HMGB3 showed that expression of HMGB3 enhanced PCa cell aggressiveness. Overexpression of HMGB3/HMGB3 was confirmed in naive PCa and castration-resistant PCa (CRPC) clinical specimens. Novel approaches to analysis of antitumor miRNA-regulated RNA networks in PCa cells may provide new insights into the pathogenic mechanisms of the disease.