Description: Purpose: Successful therapy of patients with prostate cancer is highly dependent on reliable diagnostic and prognostic biomarkers. Brachyury is considered a negative prognostic factor in colon and lung cancer; however, there are no reports on Brachyury's expression in prostate cancer. Experimental Design: In this study, we aimed to assess the impact of Brachyury expression in prostate tumorigenesis using a large series of human prostate samples comprising benign tissue, prostate intraepithelial neoplasia (PIN) lesions, localized tumor, and metastatic tissues. The results obtained were compared with what can be inferred from the Oncomine database. In addition, multiple in vitro models of prostate cancer were used to dissect the biologic role of Brachyury in prostate cancer progression. Results: We found that Brachyury is significantly overexpressed in prostate cancer and metastatic tumors when compared with normal tissues, both at protein and at mRNA levels. Brachyury expression in the cytoplasm correlates with highly aggressive tumors, whereas the presence of Brachyury in the nucleus is correlated with tumor invasion. We found that Brachyury-positive cells present higher viability, proliferation, migration, and invasion rates than Brachyury-negative cells. Microarray analysis further showed that genes co-expressed with Brachyury are clustered in oncogenic-related pathways, namely cell motility, cell-cycle regulation, and cell metabolism. Conclusions: Collectively, the present study suggests that Brachyury plays an important role in prostate cancer aggressiveness and points, for the first time, to Brachyury as a significant predictor of poor prostate cancer prognosis. Our work paves the way for future studies assessing Brachyury as a possible prostate cancer therapeutic target. Clin Cancer Res; 20(18); 4949–61. ©2014 AACR .