Matrix metalloproteinase-9 (MMP-9) displays a preference for a broad range of substrates including extracellular matrix proteins and cytokines. MMP-9 plays an important role in physiological processes, as well as in inflammatory diseases and numerous cancers. Interferon-β is a pleiotropic cytokine with antiviral, antiproliferative and immunomodulatory activities. Interferon-β positively regulates gene expression, predominantly through the Janus kinase-signal transducer and activator of transcription (STAT) pathway. However, little is known about the mechanisms used by interferon-β to negatively regulate gene expression. In the present study, we show that interferon-β inhibits MMP-9 gene expression at the transcriptional level. Using cell lines deficient in three components of the interferon-β-activated interferon-stimulated gene factor 3 (ISGF3) complex (i.e. STAT-1, STAT-2 and interferon regulatory factor 9), the results of our study indicate that all three members are required for interferon-β inhibition. Chromatin immunoprecipitation assays demonstrate that interferon-β reduces recruitment of transcriptional activators and coactivators, such as nuclear factor kappa B p65, Sp1, CREB-binding protein and p300, to the MMP-9 promoter, and decreases the degree of histone acetylation at the MMP-9 promoter. This occurs in the absence of an association of the ISGF3 complex with the MMP-9 promoter. Taken together, these data define the role of interferon-β and the ISGF3 members in suppressing MMP-9 gene expression. © 2007 The Authors.