MicroRNA-335 (miR-335) acts as a tumor suppressor or a tumor promoter in different human malignancies. However, the involvement of miR-335 in prostate cancer (PCa) is still unclear. The purpose of this study was to investigate the functional and clinical significance of miR-335 in PCa. miR-335 expression in 3 PCa cell lines (LNCaP/DU145/PC3) and in 20 clinical PCa tissues were detected by real-time quantitative reverse transcriptase-PCR compared with corresponding controls. The function of miR-335 was investigated for cell proliferation, invasion and migration in PCa cells transfected with agents containing EGFP-miR-335 expression vector. Additionally, miR-335 expression in 104 clinical PCa tissues was detected by in situ hybridization. Its assocaitions with clinicopathological features and prognosis in patients with PCa were also determined. miR-335 was significantly down-regulated in PCa cell lines than in the normal prostate cell line (P < 0.01). With the similar results in vitro, the reduced expression of miR-335 was also found in human PCa tissues comparing with paired adjacent benign prostate tissues (P < 0.05). Moreover, the increased expression of miR-335 suppressed cell proliferation, invasion and migration of PCa cell lines in vitro. Turning to its clinical significance, the low expression of miR-335 was significantly associated with high Gleason Score (P = 0.04), advanced clinical stage (P = 0.04), and positive metastasis (P = 0.02), but not with prognosis in PCa patients. Our data demonstrated for the first time the inhibitory effect of miR-335 on cell proliferation and invasion for PCa cells. The loss of this microRNA might be associated with clinical progression of PCa patients.