Abstract Prostate cancer (PCa) is the most commonly diagnosed cancer in men and the second leading cause of cancer-related deaths in the USA. Relapsed androgen signaling and intratumoral sterol biosynthesis are hallmarks of castration-resistant PCa (CRPC). PCa growth and proliferation depend on androgen signaling by the Androgen Receptor (AR). Targeting the androgen synthesis pathways and transactivation function of AR is a primary therapeutic approach to treat PCa. The progression of PCa from castration-sensitive to the castration-resistant stage (CRPC) depends on the failure of androgen signaling antagonists and the dysregulation of AR-centric molecular mechanisms in a high percentage of patients. Recent studies show that microRNAs (miRNAs) exert post-transcriptional regulation of gene expression by targeting oncogenes and performing tumor-suppressive functions in various cancers, including PCa. Scientific premise: The fundamental discovery motivating the proposed study is that miR-149-5p targets the expression of two major transcription factors AR and sterol regulatory element-binding transcription factor 1 (SREBF1), both are druggable targets and are implicated in CRPC. Our central hypothesis entails that miR-149-5p post-transcriptionally downregulates AR and SREBF1, and inactivation of miR-149-5p expression during the prostate carcinogenesis leads to the upregulation of androgen signaling and sterol biosynthesis, which promotes CRPC. We will test our hypothesis by pursuing three integrated Specific Aims. In aim 1 we will test miR-149-5p anti-proliferation and anti-oncogenic function in PCa cells, in Aim 2, we will define molecular mechanisms which negatively affects the maturation of miR-149-5p and in the aim 3, we will test the therapeutic and prognostic value of miR-149-5p. Significance: The study will provide a tumor-suppressor role of miR-149-5p in CRPC. The innovative research approaches the disease from a novel miRNA-mediated control of the androgen signaling and intratumoral sterol biosynthesis pathways in particular testosterone metabolism by directly co-targeting AR and SREBF1, and both are druggable targets in CRPC. This study has the potential to test for the first time co-targeting of primary PCa promoting transcription factors and a new paradigm for improved patient therapies by identifying the significant key regulators of androgen signaling and sterol biosynthesis pathways.