We have previously shown that lysyl oxidase (LOX) mRNA is up-regulated in invasive breast cancer cells and that catalytically active LOX facilitates in vitro cell invasion. Here we validate our in vitro studies by showing that LOX expression is up-regulated in distant metastatic breast cancer tissues compared with primary cancer tissues. To elucidate the mechanism by which LOX facilitates cell invasion, we show that catalytically active LOX regulates in vitro motility/migration and cell-matrix adhesion formation. Treatment of the invasive breast cancer cell lines, Hs578T and MDA-MB-231, with β-aminopropionitrile (βAPN), an irreversible inhibitor of LOX catalytic activity, leads to a significant decrease in cell motility/migration and adhesion formation. Conversely, poorly invasive MCF-7 cells expressing LOX (MCF-7/LOX32-His) showed an increase in migration and adhesion that was reversible with the addition of βAPN. Moreover, a decrease in activated focal adhesion kinase (FAK) and Src kinase, key proteins involved in adhesion complex turnover, was observed when invasive breast cancer cells were treated with βAPN. Additionally, FAK and Src activation was increased in MCF-7/LOX32-His cells, which was reversible on βAPN treatment. Hydrogen peroxide was produced as a by-product of LOX activity and the removal of hydrogen peroxide by catalase treatment in invasive breast cancer cells led to a dose-dependent loss in Src activation. These results suggest that LOX facilitates migration and cell-matrix adhesion formation in invasive breast cancer cells through a hydrogen peroxide–mediated mechanism involving the FAK/Src signaling pathway. These data show the need to target LOX for treatment of aggressive breast cancer. (Cancer Res 2005; 65(24): 11429-36)