Although prostate cancer (CaP) is the most frequently diagnosed malignant tumor in American men, the mechanisms underlying the development and progression of CaP remain largely unknown. Recent studies have shown that downregulation of the microRNA miR-124 occurs in several types of human cancer, suggesting a tumor suppressive function of miR-124. Until now, however, it has been unclear whether miR-124 is associated with CaP. In the present study, we completed a series of experiments to understand the functional role of miR-124 in CaP. We detected the expression level of miR-124 in clinical CaP tissues, evaluated the influence of miR-124 on the growth of CaP cells and investigated the mechanism underlying the dysregulation of miR-124. We found that (i) miR-124 directly targets the androgen receptor (AR) and subsequently induces an upregulation of p53; (ii) miR-124 is significantly downregulated in malignant prostatic cells compared to benign cells, and DNA methylation causes the reduced expression of miR-124; and (iii) miR-124 can inhibit the growth of CaP cells in vitro and in vivo. Data from this study revealed that loss of miR-124 expression is a common event in CaP, which may contribute to the pathogenesis of CaP. Our studies also suggest that miR-124 is a potential tumor suppressive gene in CaP, and restoration of miR-124 expression may represent a novel strategy for CaP therapy.