Tissue transglutaminase (TG2), an enzyme involved in cell proliferation, differentiation and apoptosis is overexpressed in ovarian carcinomas, where it modulates epithelial-to-mesenchymal transition (EMT) and promotes metastasis. Its regulation in ovarian cancer (OC) remains unexplored. Here, we show that transforming growth factor (TGF)-Β, a cytokine involved in tumor dissemination is abundantly secreted in the OC microenvironment and induces TG2 expression and enzymatic activity. This is mediated at transcriptional level by SMADs and by TGF-Β-activated kinase 1-mediated activation of the nuclear factor-B complex. TGF-Β-stimulated OC cells aggregate as spheroids, which enable peritoneal dissemination. We show that TGF-Β-induced TG2 regulates EMT, formation of spheroids and OC metastasis. TG2 knock-down in OC cells decreases the number of cells harboring a cancer stem cell phenotype (CD44 +CD117+). Furthermore, CD44 +CD117+ cells isolated from human ovarian tumors express high levels of TG2. In summary, TGF-Β-induced TG2 enhances ovarian tumor metastasis by inducing EMT and a cancer stem cell phenotype. © 2012 Macmillan Publishers Limited All rights reserved.