Background: Prostate cancer (PCa) is a major cause of morbidity and mortality in men worldwide. MicroRNAs are globally downregulated in PCa, especially in poorly differentiated tumors. Nonetheless, the underlying mechanisms are still elusive. Herein, using combined analysis of microRNAs expression and genomewide DNA methylation, we aimed to identify epigenetically downregulated microRNAs in PCa. Results: We found that miR-152-3p was underexpressed in PCa and that lower expression levels were associated with promoter hypermethylation in accordance with TCGA dataset analysis. Functional in vitro assays suggest that miR-152-3p suppresses cell viability and invasion potential, whereas it promotes cell cycle arrest at S and G2/M phases. Additionally, miR-152-3p expression was associated with longer disease-free survival in PCa patients from TCGA. Finally, TMEM97, which is overexpressed in PCa, was identified as a novel miR-152-3p target gene. Conclusions: Our findings demonstrate the advantages of using a combinatory approach to identify microRNAs downregulated due to aberrant promoter methylation. MiR-152-3p downregulation and promoter methylation was found to be prevalent in primary PCa, which impairs its role in control of cell viability, cell cycle regulation and invasion.