FAT1 [Homo sapiens FAT tumor suppressor homolog 1 (Drosophila)] is an intrinsic membrane protein classified as a member of the cadherin superfamily. The FAT1 gene is a tumor suppressor in humans as well as being the pivotal gene for cell morphogenesis and migration. Deletion of this gene could play a role in the characteristics of oral squamous cell carcinomas (OSCCs), involving cell adhesion, migration and/or invasion. This study investigated the mechanisms by which FAT1 is involved in the biological behavior of OSCCs. First, a rat monoclonal antibody was developed against a FAT1 intra-cellular domain epitope, and used for an immunohistochemical study of FAT1 in clinically obtained OSCC samples. FAT1 was localized at lamellipodial edges or cell-cell boundaries in normal cells and well differentiated OSCCs, but showed a diffuse cytoplasmic and nuclear distribution in moderately-poorly differentiated OSCCs. FAT1-siRNA was transfected into OSCCs resulting in a drastic inhibition of cell migration and invasion based on the suppression of FAT1 expression and disorganized localization of β-catenin which is associated with cell polarity and migration. These results suggested that FAT1 may be involved in the migration and invasion mechanisms of OSCCs and, therefore, it could be an important target for the development of new therapeutic strategies.