Limited information is available on the pathologic significance of human antigen R (HuR) in prostate cancer (PCa). The main aim of this study was to clarify the relationship between HuR expression and malignant aggressiveness, outcome, and expression of cancer-related molecules in PCa. In vitro proliferation, colony formation, and migration assays were performed on LNCaP and PC-3 cells. HuR expression was knocked down (KD) using small interfering RNA. The relationships between HuR expression and the expression of vascular endothelial growth factors (VEGFs), cyclooxygenase (COX)-2, and heme oxygenase (HO)-1 were investigated in PCa cell lines using Western blotting. On KD of HuR, cell proliferation and migration were suppressed in both LNCaP and PC-3 cells, whereas expression of VEGF-A to -D and COX-2 was suppressed in PC-3 but not in LNCaP cells. In addition, expression of these cancer-related factors was analyzed in 182 hormone-naïve PCa and 23 castration-resistant prostate cancer (CRPC) human tissues in vivo. Cytoplasmic (C)-HuR expression was significantly higher in CRPC > hormone-naïve PCa > nontumoral cells. C-HuR expression was positively associated with Gleason score, T stage, and metastasis, and it was considered to be a useful predictor of biochemical recurrence after radical prostatectomy. C-HuR expression was correlated with COX-2 expression in hormone-naïve PCa, and with the expression of VEGF-A, VEGF-C, and COX-2 in CRPC tissues. Our results demonstrated that HuR plays important roles in determining malignant aggressiveness and outcome in PCa, especially in androgen-independent PCa cells, via the regulation of cell proliferation, migration, and expression of VEGF-A, -C, and COX-2.