Prostate cancer is one of the leading causes of cancer deaths among men, many miRNAs have been demonstrated to play critical role in the progression of prostate cancer, miR-186 suppresses the progression of many tumors, such as bladder cancer and glioma. Previous study shows miR-186 is downregulated in prostate cancer tissues, and is a good prognosis for prostate cancer patients. In this study, we found miR-186 was downregulated in prostate cancer cells and tissues, overexpression of miR-186 inhibited cell proliferation and tumorigenesis in vitro determined by MTT assay, colony formation assay and soft agar growth assay, whereas knockdown of miR-186 reduced these effects. Cell cycle analysis found miR-186 overexpression arrested cell cycle in G0/G1 phase, and reduced p21 and p27 levels, and enhanced Cyclin D1 and the phosphorylation of Rb levels, suggesting miR-186 blocked G1/S transition. A novel oncogene Golgi phhosphoprotein 3 (GOLPH3) was the target of miR-186, miR-186 bound to the 3'UTR of GOLPH3. Moreover, miR-186 was negatively correlated with GOLPH3 in prostate cancer tissues. In conclusion, our study suggested miR-186 inhibited cell proliferation through targeting oncogene GOLPH3.