MacroD2 is one of the three human macrodomain proteins characterized by their protein-linked mono-ADP-ribosyl-hydrolyzing activity. MacroD2 is a single-domain protein that contains a deep ADP-ribose-binding groove. In this study, new crystallization conditions for MacroD2 were found and three crystal structures of human MacroD2 in the apo state were solved in space groupsP4₁2₁2,P4₃2₁2 andP4₃, and refined at 1.75, 1.90 and 1.70 Å resolution, respectively. Structural comparison of the apo crystal structures with the previously reported crystal structure of MacroD2 in complex with ADP-ribose revealed conformational changes in the side chains of Val101, Ile189 and Phe224 induced by the binding of ADP-ribose in the active site. These conformational variations may potentially facilitate design efforts of a MacroD2 inhibitor.