Background Herein, we identified miRNA signatures that were able to differentiate malignant prostate cancer from benign prostate hyperplasia and revealed the therapeutic potential of these miRNAs against prostate cancer development. Methods and results MicroRNA expressions were determined by qPCR. MTT was used for cell viability analysis and immunohistochemistry was performed for Bax/Bcl-2 staining. ELISA was used to measure MMP2/9 levels. Wound healing assay was used for the evaluation of cell migration. Notably, expression levels of miR-125b-5p, miR-145-5p and miR-221-3p were significantly reduced in prostate cancer patients as compared to BPH patients. Moreover, ectopic expression of miR-125b-5p, miR-145-5p and miR-221-3p resulted in significant inhibition of cell proliferation and altered cell morphology. Also, expression level of Bax protein was increased while Bcl-2 level was reduced in cells treated with miR-125b-5p, miR-145-5p and miR-221-3p mimics. Enhanced expression of miR-125b-5p, miR-145-5p and miR-221-3p was also significantly altered the expression of caspase 3 and 8 levels. In addition, MMP9 levels were significantly reduced in cells ectopically expressing miR-221-3p. All miRNA mimics significantly interfered with the migration of prostate cancer cells. Conclusions Consequently, our findings point to an important role of these three miRNAs in prostate cancer and indicate that miR-125b-5p, miR-145-5p and miR-221-3p are potential therapeutic targets against prostate cancer.