Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is involved in several biological processes including cell adhesion, invasion, and angiogenesis. HB-EGF also plays a pivotal role in the progression of ovarian cancer. To investigate the significance of HB-EGF in peritoneal dissemination, we examined the roles of HB-EGF in cell adhesion, invasion, and angiogenesis in ovarian cancer. Through the suppression of focal adhesion kinase and EGF receptor activation, cell adhesive properties mediated by integrin beta(1) were diminished by the inhibition of HB-EGF expression. The reduction of HB-EGF expression attenuated the chemotactic invasive ability and the expression of matrix metalloprotease (MMP)-2 and vascular endothelial growth factor (VEGF), leading to the inhibition of cell invasion and angiogenesis. Suppression of the Snail family, which regulates the epithelial-mesenchymal transition, blocked the cell adhesion properties on extracellular matrices, the chemotactic invasive ability, and the expression of MMP9 and VEGF through the reduction of HB-EGF expression. The volume of tumor burden in the peritoneal cavity was dependent on the expression of HB-EGF. According to these results, HB-EGF contributes to cell adhesion, invasion, and angiogenesis, which are integral to transcoelomic metastasis in ovarian cancer. CRM197, an inhibitor of HB-EGF, resulted in a significant decrease of tumor burden in peritoneal dissemination, accompanied with a reduction in both cellular spreading, when assayed on an extracellular matrix, and invasive ability, when assayed in a chemotaxis chamber, as well as decreased expression of MMP9 and VEGF. Thus, HB-EGF is a mutual validating target in the peritoneal dissemination of ovarian cancer, and CRM197 may be useful as a anticancer agent for advanced ovarian cancer.