Kallikrein-related peptidase 7 (KLK7) is upregulated in epithelial ovarian carcinoma (EOC) with high levels correlated with poor prognosis. However, the mechanisms underlying this relationship and the role of KLK7 in EOC progression are unknown. We report that two different KLK7 transcripts, KLK7-253 and KLK7-181, are simultaneously expressed in high-grade serous EOC. Multicellular aggregates (MCA), which promote cell survival and chemoresistance, were observed in SKOV-3 cells stably overexpressing KLK7-253 in particular. Importantly, these MCAs invade into a monolayer of mesothelial cells and form cancer cell foci. Blocking MCA using antibodies against KLK7 and alpha(5)beta(1) and beta(1) integrins confirmed the involvement of KLK7 and integrin-regulated cell adhesion. Increased levels of alpha(5)/beta(1) integrins and enhanced attachment to fibronectin and vitronectin, which was blocked with an anti-beta(1) integrin antibody, were also observed. Finally, Western blot and immunohistochemistry showed higher KLK7 and alpha(5)/beta(1) integrin levels in serous EOC cells from ascites and tumor samples from chemotherapy nonresponders with short postsurvival times. Additionally, both KLK7-253 and KLK7-181 clones were more resistant to paclitaxel treatment in vitro. These findings suggest a mechanism for the association of high KLK7 levels with chemoresistance and poor prognosis for serous EOC patients by promotion of peritoneal dissemination and reinvasion via increased MCA and alpha(5)beta(1) integrin-dependent cell adhesion.