Natalizumab antibody to ?4-integrins is used in therapy of multiple sclerosis and Crohn's disease. A crystal structure of the Fab bound to an ?4 integrin ?-propeller and thigh domain fragment shows that natalizumab recognizes human-mouse differences on the circumference of the ?-propeller domain. The epitope is adjacent to but outside of a ligand-binding groove formed at the interface with the ?-subunit ?I domain and shows no difference in structure when bound to Fab. Competition between Fab and the ligand vascular cell adhesion molecule (VCAM) for binding to cell surface ?4?1 shows noncompetitive antagonism. In agreement, VCAM docking models suggest that binding of domain 1 of VCAM to ?4-integrins is unimpeded by the Fab, and that bound Fab requires a change in orientation between domains 1 and 2 of VCAM for binding to ?4?1. Mapping of species-specific differences onto ?4?1 and ?4?7 shows that their ligand-binding sites are highly conserved. Skewing away from these conserved regions of the epitopes recognized by current therapeutic function-blocking antibodies has resulted in previously unanticipated mechanisms of action.