MicroRNAs are a class of naturally occurring small non-coding RNAs that target protein-coding mRNAs at the post-transcriptional level and regulate complex patterns of gene expression. Our previous studies demonstrated that in human prostate cancer the miRNA miR-125b is highly expressed, leading to a negative regulation of some tumor suppressor genes. In this study, we further extend our studies by showing that miR-125b represses the protein product of the ink4a/ARF locus, p14(ARF), in two prostate cancer cell lines, LNCaP (wild type-p53) and 22Rv1 (both wild type and mutant p53), as well as in the PC-346C prostate cancer xenograft model that lentivirally overexpressed miR-125b. Our results highlight that miR-125b modulates the p53 network by hindering the down-regulation of Mdm2, thereby affecting p53 and its target genes p21 and Puma to a degree sufficient to inhibit apoptosis. Conversely, treatment of prostate cancer cells with an inhibitor of miR-125b (anti-miR-125b) resulted in increased expression of p14(ARF), decreased level of Mdm2, and induction of apoptosis. In addition, overexpression of miR-125b in p53-deficient PC3 cells induced down-regulation of p14(ARF), which leads to increased cell proliferation through a p53-independent manner. Thus, we conclude that miR-125b acts as an oncogene which regulates p14(ARF)/Mdm2 signaling, stimulating proliferation of prostate cancer cells through a p53-dependent or p53-independent function. This reinforces our belief that miR-125b has potential as a therapeutic target for the management of patients with metastatic prostate cancer.