Abelson interactor protein-1 (ABI-1) is an adaptor protein involved in actin reorganization and lamellipodia formation. It forms a macromolecular complex containing Hspc300/WASP family verprolin-homologous proteins 2/ABI-1/nucleosome assembly protein 1/PIR121 or Abl/ABI-1/WASP family verprolin-homologous proteins 2 in response to Rho family-dependent stimuli. Due to its role in cell mobility, we hypothesized that ABI-1 has a role in invasion and metastasis. In the present study, we found that weakly invasive breast cancer cell lines (MCF-7, T47D, MDA-MB-468, SKBR3, and CAMA1) express lower levels of ABI-1 compared with highly invasive breast cancer cell lines (MDA-MB-231, MDA-MB-157, BT549, and Hs578T), which exhibit high ABI-1 levels. Using RNA interference, ABI-1 was stably down-regulated in MDA-MB-231, which resulted in decreased cell proliferation and anchorage-dependent colony formation and abrogation of lamellipodia formation on fibronectin. Down-regulation of ABI-1 decreased invasiveness and migration ability and decreased adhesion on collagen IV and actin polymerization in MDA-MB-231 cells. Additionally, compared with control parental cells, ABI-1 small interfering RNA-transfected cells showed decreased levels of phospho-PDK1, phospho-Raf, phospho-AKT, total AKT, and AKT1. These data suggest that ABI-1 plays an important role in the spread of breast cancer and that this role may be mediated via the phosphatidylinositol 3-kinase pathway.