Our recent study of microRNA (miRNA) expression signatures in prostate cancer (PCa) has revealed that all members of the miR-23b/27b/24-1 cluster are significantly downregulated in PCa tissues. The aim of this study was to investigate the effectiveness of these clustered miRNAs as a disease progression marker and to determine the functional significance of these clustered miRNAs in PCa. Expression of the miR-23b/27b/24-1 cluster was significantly reduced in PCa tissues. Kaplan-Meier survival curves showed that low expression of miR-27b predicted a short duration of progression to castration-resistant PCa. Gain-of-function studies using mature miR-23b, miR-27b,and miR-24-1 significantly inhibited cell proliferation, migration and invasion in PCa cells (PC3 and DU145). To identify the molecular targets of these miRNAs, we carried out gene expression and in silico database analyses. GOLM1 was directly regulated by miR-27b in PCa cells. Elucidation of the molecular targets and pathways regulated by the tumor-suppressive microRNAs should shed light on the oncogenic and metastatic processes in PCa.