Interferon-β (IFNβ) therapy is effective in approximately half of the patients with relapsing-remitting multiple sclerosis (RRMS). Clinical non-responders were characterized by an increased expression of IFN response genes before the start of therapy, and a lack of a pharmacologically induced increase in IFN response gene activity. Because Interferon Regulatory Factor 5 (IRF5) is a master regulator of IFN-activity, we carried out a candidate gene study of IRF5 gene variants in relation to the pharmacological and clinical response upon IFNβ treatment. We found that patients with the IRF5 rs2004640-TT and rs47281420-AA genotype exerted a poor pharmacological response to IFNβ compared with patients carrying the respective G-alleles (P= 0.0006 and P= 0.0023, respectively). Moreover, patients with the rs2004640-TT genotype developed more magnetic resonance imaging (MRI)-based T2 lesions during IFNβ treatment (P= 0.003). Accordingly, an association between MRI-based non-responder status and rs2004640-TT genotype was observed (P= 0.010). For the rs4728142-AA genotype a trend of an association with more T2 lesions during IFNβ treatment and MRI-based non-responder status was observed (P= 0.103 and P= 0.154, respectively). The clinical relevance of the rs2004640-TT genotype was validated in an independent cohort wherein a shorter time to first relapse was found (P= 0.037). These findings suggest a role for IRF5 gene variation in the pharmacological and clinical outcome of IFNβ therapy that might have relevance as biomarker to predict the response to IFNβ in multiple sclerosis.