HLA‐DO (DO) is a non‐polymorphic, non‐classical MHC class II that is known to inhibit MHC class II peptide loading mediated by DM. The mechanism of inhibition by DO as well as the interactions between DO and DM remain unknown. Here, we present the 3D structure of the DM and DO complex determined by x‐ray crystallography at 3.2 Å resolution which is also the first resolved structure of DO. DO exhibits close structural similarity to the MHC class II molecule. The crystal complex shows DO residues mainly at the N‐terminal side of the α1 peptide‐binding groove interact with DM residues located at a concave surface. Some interactions are also observed involving DO residues from the IgG‐like domain. DM concave surface includes an acidic patch where residues in and surrounding this acidic patch and residues in the IgG‐like domain interact with DO. Interestingly, the interaction surface on DM to DO is spatially close to DM's interaction surface for DR that was suggested by molecular modeling and mutagenesis studies. The novel structure of the DM/DO complex presented here provides an insight on the possible mechanism of inhibition of DO on DM as well as the mechanism of how DM catalyzes peptide loading to peptide free DR.