The formation of antibodies to interferon-beta (IFN-β), a protein-based disease-modifying agent for multiple sclerosis (MS), is a problem in clinical practice. These antibodies may neutralize the biological effects of the protein drug, potentially decreasing its therapeutic effects. By high-resolution HLA class I and II typing we identified two HLA class II alleles associated with the development of antibodies to IFN-β. In two independent continuous and binary-trait association studies, HLA-DRB1^∗0401 and HLA-DRB1^∗0408 (odds ratio: 5.15)—but not other HLA alleles—were strongly associated with the development of binding and neutralizing antibodies to IFN-β. The associated HLA-DRB1^∗04 alleles differ from nonassociated HLA-DRB1^∗04 alleles by a glycine-to-valine substitution in position 86 of the epitope-binding alpha-helix of the HLA class II molecule. The peptide-binding motif of HLA-DRB1^∗0401 and ^∗0408 might promote binding and presentation of an immunogenic peptide, which may eventually break T cell tolerance and facilitate antibody development to IFN-β. In summary, we identified genetic factors determining the immunogenicity of IFN-β, a protein-based disease-modifying agent for the treatment of MS.