It has been reported that one of the factors that promotes tumoral progression is the abnormal activation of the epithelial-mesenchymal transition program. This process is associated with tumoral cells acquiring invasive and malignant properties and has the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1) as one of its main activators. However, the role of ZEB1 in promoting malignancy in prostate cancer (PCa) is still unclear. Here, we report that ZEB1 expression correlates with Gleason score in PCa samples and that expression of ZEB1 regulates epithelial-mesenchymal transition and malignant characteristics in PCa cell lines. The results showed that ZEB1 expression is higher in samples of higher malignancy and that overexpression of ZEB1 was able to induce epithelial-mesenchymal transition by upregulating the mesenchymal marker Vimentin and downregulating the epithelial marker E-Cadherin. On the contrary, ZEB1 silencing repressed Vimentin expression and upregulated E-Cadherin. ZEB1 expression conferred enhanced motility and invasiveness and a higher colony formation capacity to 22Rv1 cells whereas DU145 cells with ZEB1 silencing showed a decrease in those same properties. The results showed that ZEB1 could be a key promoter of tumoral progression toward advanced stages of PCa.