OBJECTIVE: MiR-564 has been discovered to be abnormally expressed in human malignancy. Two recent studies suggested that miR-564 plays a role in tumor inhibition in both lung and breast cancer. However, no evidence reported the mechanism and function of miR-564 in prostate cancer (PCa). PATIENTS AND METHODS: The PCa tissues and their adjacent normal tissues were collected from 50 PCa patients. Expressions of miR-564 in tissues and cells were evaluated with RT-qPCR. The MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide] assay, ?ow cytometry and Western-blot analysis, were applied to detect the proliferation, cell cycle progression and the protein expression of PCa cell lines (PC-3 and DU-145). Migration and invasion of PCa cells were analyzed by Transwell assays. Furthermore, the correlation between miR-564 and MLLT3 was assessed by luciferase reporter assay. Also, the PCa cells were transfected with miR-564 mimics control and inhibitor. RESULTS: In our present research, miR-564 was found dysregulated in PCa cells and to act as a suppressor in PCa cell proliferation, progression of cell cycle, cell invasion and migration. MLLT3 (also known as Af9) is a proto-oncogene, which has first reported in leukemia, and the regulation of its expression remains incompletely elucidated. Also, it is first reported in our study, suggesting that MLLT3 is a direct target of miR-564. The results also showed a significant negative correlation with miR-564 in PCa cells. Furthermore, up-regulation of MLLT3 attenuates the effects of miR-564 on the ability of PCa cells. CONCLUSIONS: Our research demonstrated the suppressor function of miR-564 in PCa, revealing restoration of miR-564 as a potential therapeutic strategy for the treatment of PCa.