MicroRNAs (miRs) are small non‑coding RNA molecules that regulate gene expression at the post‑transcriptional level. Aberrant expression of miR‑9 has been reported to be involved in the tumorigenesis and progression of various malignancies. However, its role in prostate cancer (PC) has not been completely clarified. In the present study, miR‑9 expression was examined in different PC cell lines, patient tissues and a mouse model. Cell Counting Kit‑8 and BrdU immunofluorescence assays were performed to assess the effect of miR‑9 on the viability of PC cells, while Transwell and wound‑healing assays were utilized to evaluate the migration and invasion of PC cells expressing miR‑9. Furthermore, a dual‑luciferase reporter assay was performed to verify whether mitogen‑activated protein kinase kinase kinase 3 (MEKK3) was a direct target of miR‑9. The results demonstrated significant downregulation of miR‑9 expression in different PC cell lines and 31 human PC tissues, as compared with that in a normal prostate cell line and adjacent normal tissues, respectively. By contrast, upregulation of MEKK3 was confirmed in human PC tissue samples, with its level inversely associated with miR‑9 expression. Overexpression of miR‑9 in six different PC cell lines (DU145, LNCaP, 22Rv1, PC‑3, C4‑2B and VCaP) reduced the cell viability and migration. Furthermore, it was demonstrated that the 3'‑untranslated region of MEKK3 was a target of miR‑9, and that MEKK3 overexpression prevented the inhibitory effects of miR‑9 on the viability, migration and invasion of PC cells. miR‑9 overexpressing tumor cells also exhibited growth delay in comparison with control tumor cells in vivo. Taken together, the current study findings provided novel insights into the underlying molecular mechanisms of PC oncogenesis, which may support the development of new therapeutic approaches for the treatment of PC.