Atomic force microscopy (AFM) has been a useful technique to visualize cellular and molecular structures at single-molecule resolution. The combination of imaging and force modes has also allowed the characterization of physical properties of biological macromolecules in relation to their structures. Furthermore, recognition imaging, which is obtained under the TREC(TM) (Topography and RECognition) mode of AFM, can map a specific protein of interest within an AFM image. In this study, we first demonstrated structural properties of purified α Actinin-4 by conventional AFM. Since this molecule is an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction, we investigated their structural properties using the recognition mode of AFM. For this purpose, we attached an anti-α Actinin-4 monoclonal antibody to the AFM cantilever and performed recognition imaging against α Actinin-4. We finally succeeded in mapping the epitopic region within the α Actinin-4 molecule. Thus, recognition imaging using an antibody coupled AFM cantilever will be useful for single-molecule anatomy of biological macromolecules and structures.