Histone deacetylase 6 (HDAC6) is an emerging target for the treatment of various diseases such as cancer through selective inhibition. While the second catalytic domain (HDAC6 CD2) has been extensively studied, comparatively little research has focused on the first catalytic domain (HDAC6 CD1). This work provides new structural insights regarding inhibitor binding to HDAC6 CD1 enabled by the study of active-site mutants.