The αvβ6 integrin is up-regulated in cancer and wound healing but it is not generally expressed in healthy adult tissue. There is increasing evidence that it has a role in cancer progression and will be a useful target for antibody-directed cancer therapies. We report a novel recombinant diabody antibody fragment that targets specifically αvβ6 and blocks its function. The diabody was engineered with a C-terminal hexahistidine tag (His tag)， expressed in Pichia pastoris and purified by IMAC. Surface plasmon resonance (SPR) analysis of the purified diabody showed affinity in the nanomolar range. Pre-treatment of αvβ6-expressing cells with the diabody resulted in a reduction of cell migration and adhesion to LAP， demonstrating biological function-blocking activity. After radio-labeling， using the His-tag for site-specific attachment of (99m)Tc， the diabody retained affinity and targeted specifically to αvβ6-expressing tumors in mice bearing isogenic αvβ6 +/- xenografts. Furthermore， the diabody was specifically internalized into αvβ6-expressing cells， indicating warhead targeting potential. Our results indicate that the new αvβ6 diabody has a range of potential applications in imaging， function blocking or targeted delivery/internalization of therapeutic agents.