OBJECTIVE: The aim of this study was to investigate the inhibitory role of microRNA-1299 (miR-1299) in prostate cancer, and to explore the possible underlying mechanism. PATIENTS AND METHODS: The expression of miR-1299 in 35 PCa tissues and pars-carcinoma tissues, as well as PCa cell lines (PC-3) and prostatic epithelial cell line (RWPE-1). was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Then, we explored the possible targets of miR-1299 by searching online databases. NIMA-related kinase 2 (NEK2) was identified as a direct target gene of miR-1299. Subsequently, qRT-PCR. Western blot (WB), and luciferase reporter gene assay were used to further verify the correlation between miR-1299 and NEK2. To better characterize the role of miR-1299 and NEK2 in PCa, we conducted functional experiments (MTT, flow cytometry, scratch-wound, and transwell assay) by transfecting PC-3 cells with miR-1299 mimics and si-NEK2 in different groups. RESULTS: The expression level of miR-1299 in PCa tissues was significantly lower than that of para-carcinoma tissues. Meanwhile, the expression of miR-1299 in PC-3 cells was also significantly downregulated when compared with RWPE-1 cells. Subsequent qRT-PCR, WB, and luciferase reporter gene assay verified that miR-1299 transcriptionally repressed NEK2 by interacting with the essential binding sequence in 3'UTR. Also, functional experiments demonstrated that decreased expression of NEK2 resulting from miR-1299 up-regulation could remarkably inhibit the proliferation, invasion, and migration of PCa cells. CONCLUSIONS: Our study indicated that miR-1299 was a novel suppressor in PCa through its negative regulation of NEK2. Moreover, our findings revealed that miR-1299/NEK2 axis might be a potential therapeutic target for the treatment of PCa.