Relapsed prostate cancer (CaP), usually treated with androgen deprivation therapy, acquires resistance to develop into lethal metastatic castration-resistant CaP. The cause of resistance remains elusive, and the lack of biomarkers predictive of castration-resistance emergence is a stumbling block in managing the disease. We provide strong evidence that Myeloid differentiation factor-2 (MD2) plays a critical role in metastasis and CaP progression.Analysis of tumor genomic data and IHC of tumors showed a high frequency ofMD2amplification and association with poor overall survival in patients. The Decipher-genomic test validated the potential ofMD2in predicting metastasis.In vitrostudies demonstrated that MD2 confers invasiveness by activating MAPK and NF-kB signaling pathways. Furthermore, we show that metastatic cells release MD2 (sMD2). We measured serum-sMD2 in patients and found that the level is correlated to disease extent. We determined the significance of MD2 as a therapeutic target and found that targeting MD2 significantly inhibited metastasis in a murine model.We conclude that MD2 predicts metastatic behavior and serum-MD2 is a non-invasive biomarker for tumor burden, whereas MD2 presence on prostate biopsy predicts adverse disease outcome. We suggest MD2-targeted therapies could be developed as potential treatments for aggressive metastatic disease.