Interferon-β (IFN-β) is one of the major drugs for multiple sclerosis (MS) treatment. The purpose of this study was to characterize the transcriptional effects induced by intramuscular IFN-β-1a therapy in patients with relapsing-remitting form of MS. By using Affymetrix DNA microarrays, we obtained genome-wide expression profiles of peripheral blood mononuclear cells of 24 MS patients within the first 4 weeks of IFN-β administration. We identified 121 genes that were significantly up- or downregulated compared with baseline, with stronger changed expression at 1 week after start of therapy. Eleven transcription factor-binding sites (TFBS) are overrepresented in the regulatory regions of these genes, including those of IFN regulatory factors and NF-κB. We then applied TFBS-integrating least angle regression, a novel integrative algorithm for deriving gene regulatory networks from gene expression data and TFBS information, to reconstruct the underlying network of molecular interactions. An NF-κB-centered sub-network of genes was highly expressed in patients with IFN-β-related side effects. Expression alterations were confirmed by real-time PCR and literature mining was applied to evaluate network inference accuracy.