MicroRNAs (miRNAs) are known to serve a role in tumorigenic programs. The dysregulated expression of miR-301a-3p may affect the progression of various types of human cancer; however, the expression and the role of miR-301a-3p in prostate cancer are still unclear. The present study aimed to clarify the role and molecular mechanism of miR-301a-3p in prostate cancer. The results demonstrated that the expression of miR-301a-3p was significantly upregulated in human prostate cancer tissues and in several prostate cancer cell lines. In vitro overexpression of miR-301a-3p notably increased prostate cancer cell proliferation and invasion. Bioinformatics analysis revealed that runt-related transcription factor 3 (RUNX3) may be a target of miR-301a-3p, which was confirmed by Dual-luciferase reporter assay. Western blot analysis also demonstrated that miR-301a-3p regulated the protein expression levels of RUNX3. In addition, the results indicated that miR-301a-3p may regulate the Wnt signaling pathway, and rescue experiments indicated that RUNX3 contributed to the effects of miR-301a-3p on cell proliferation and invasion through Wnt signaling. In conclusion, these findings suggested that miR-301a-3p may promote prostate cancer cell invasion and proliferation by targeting RUNX3, and provided insight into understanding prostate cancer pathogenesis. miR-301a-3p may be a potential therapeutic candidate to treat prostate cancer.