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MicroRNA served as inhibitor for gene expression in various cancers. This study aimed to investigate the role of miR-605 and EN2 in prostate cancer (PCa).In this research, the expression of miR-605 and EN2 protein in PCa tissues and cells were determined by qRT-PCR and western blot, respectively. The cell proliferation was measured by Cell Counting Kit-8 (CCK-8) and the tumor cell invasion assay was accomplished with transwell system. Flow cytometry was used to analyze the cell cycle. The endogenous expression of miR-605 and EN2 was modulated by recombinant plasmids and cell transfection. Dual luciferase reporter assay was performed to determine the interaction between miR-605 and EN2 in PCa cells.The expression of miR-605 was lower in PCa tissue and cells than that in normal tissues and cells, while the expression of EN2 was just the opposite. Down-regulation of the EN2 by siRNA inhibited the proliferation and invasion of PC3 cells, and the cell cycle was arrested in G0/G1 phase. EN2 regulated the expression of E-cadherin and Vimentin through Snail and EN2 regulated the cell cycle and cell proliferation via PI3K/AKT pathway. MiR-605 inhibited the proliferation and invasion of PCa cells through targeting EN2.EN2 is negatively regulated by miR-605, and down-regulation of miR-605 promotes the proliferation and invasion of PCa cells by up-regulating EN2, which leads to PCa development and progression.