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Prostate cancer (PCa) exhibits a high incidence among men, but there is no effective and non-invasive biomarker for the diagnosis of PCa, and the pathogenesis of PCa remains unclear. The present study identified that miR-27a was significantly overexpressed in the tumor tissues and sera of patients with PCa. In addition, high serum levels of miR-27a were correlated with poor survival in patients with PCa. Receiver-operating characteristic curves analysis demonstrated that the serum levels of miR-27a exhibited a high area under the curve value. Furthermore, miR-27a mimics or inhibitors significantly promoted or repressed the proliferation of PCa cells, respectively. In addition, it was identified that the expression of Sprouty2 (SPRY2) was inversely correlated with the expression of miR-27a in PCa tissues. The knockdown or overexpression of SPRY2 promoted or suppressed the proliferation of PCa cells, respectively, and the overexpression of SPRY2 inhibited the increased proliferation and cell cycle distribution of PCa cells mediated by miR-27a mimics. Taken together, these data indicated that the serum levels of miR-27a may be a novel and non-invasive biomarker for the diagnosis and prognosis of patients with PCa, and miR-27a/SPRY2 may be a therapeutic target for the treatment of PCa.