Updated project metadata. Prostate cancer (PCa) continues to be one of the most common cancers in men worldwide. The six transmembrane epithelial antigen of the prostate 1 (STEAP1) protein is overexpressed in several types of human tumours, particularly in PCa. Our research group has demonstrated that STEAP1 overexpression is associated with PCa progression and aggressiveness. Therefore, understanding the cellular and molecular mechanisms triggered by STEAP1 overexpression will provide important insights to delineate new strategies for PCa treatment. In the present work, a proteomic strategy was used to characterize the intracellular signalling pathways and the molecular targets downstream of STEAP1 in PCa cells. A label-free approach was applied using an Orbitrap LC-MS/MS system to characterize the proteome of STEAP1-knockdown PCa cells. More than 6700 proteins were identified, of which a total of 526 proteins were found differentially expressed in scramble siRNA versus STEAP1 siRNA (234 proteins up-regulated and 292 proteins down-regulated). Bioinformatics analysis allowed us to explore the mechanism through which STEAP1 exerts influence on PCa, revelling that endocytosis, RNA transport, apoptosis, aminoacyl-tRNA biosynthesis and metabolic pathways are the biological processes where STEAP1 is involved. By immunoblotting, it was confirmed that STEAP1 knockdown induced the up-regulation of cathepsin B, intersectin-1 and syntaxin 4,while it down-regulation HRas, PIK3C2A and DIS3. These finding suggested that blocking STEAP1 might be a strategy to activate apoptosis and endocytosis, and diminish cellular metabolism and intercellular communication, leading to inhibition of PCa progression.