microRNAs (miRNAs) are known to be important in tumor initiation and progression. Recent studies have demonstrated that miR-186 is critical in several types of cancer, including human non-small cell lung cancer, bladder cancer and pancreatic ductal adenocarcinoma. However, the functions of miR-186 in prostate cancer (PCa) are still unclear. In the present study, downregulation of miR-186 in PCa cells was detected when compared with the normal prostate cell line. When miR-186 overexpressed in PCa cells, cell proliferation in vitro was evidently inhibited as shown using cell counting kit-8 assays and cell-cycle analysis, and tumor growth in vivo was decreased as shown by tumor growth assays in nude mice. Furthermore, through bioinformatics prediction and biochemical analyses, Yin Yang 1 (YY1) and cyclin-dependent kinase 6 (CDK6) have been proven to act as direct targets of miR-186. These results indicate that miR-186 is a negative regulator in PCa by inhibiting PCa cell proliferation via targeting YY1 and CDK6.