Background: Previous studies report that miR-1-3p, a member of the microRNA-1 family (miR-1), and functions as a tumor suppressor in several different cancers. However, little is known regarding the biological role and intrinsic regulatory mechanisms of miR-1-3p in prostate cancer (PCa). Methods: In this study, the expression levels of miR-1-3p were first examined in PCa cell lines and tumor tissues by RT-qPCR and bioinformatics. The in vitro and in vivo functional effect of miR-1-3p was examined further. A luciferase reporter assay was conducted to confirm target associations. Results: We found that miR-1-3p was significantly downregulated in advanced PCa tissues and cell lines. Low miR-1-3p levels were strongly associated with aggressive clinicopathological features and poor prognosis in PCa patients. Ectopic expression of miR-1-3p in 22RV1 and LncaP cells was sufficient to prevent tumor cell growth and cell cycle progression in vitro and in vivo. Further mechanistic studies revealed that miR-1-3p could directly target the mRNA 3'- untranslated region (3'- UTR) of two central cell cycle genes, E2F5 and PFTK1, and could suppress their mRNA and protein expression. In addition, knockdown of E2F5 and PFTK1 mimicked the tumor-suppressive effects of miR-1-3p overexpression on PCa progression. Conversely, concomitant knockdown of miR-1-3p and E2F5 and PFTK1 substantially reversed the inhibitory effects of either E2F5 or PFTK1 silencing alone. Conclusion: These data highlight an important role for miR-1-3p in the regulation of proliferation and cell cycle in the molecular etiology of PCa and indicate the potential for miR-1-3p in applications furthering PCa prognostics and therapeutics.