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.