Interferon-γ (IFN-γ) is a pleiotropic cytokine involved in aspects of immune regulation, cell proliferation, and host defense mechanisms directed toward various cancers. Some of the biological functions of IFN-γ are achieved through inhibition of gene expression, although the mechanisms by which IFN-γ suppresses gene transcription are poorly understood. Herein, we demonstrate the molecular basis by which IFN-γ mediates suppression of the matrix metalloproteinase-9 (MMP-9) gene. IFN-γ-activated signal transducer and activator of transcription-1α (STAT-1α) suppresses MMP-9 gene transcription, which is dependent on phosphorylation of tyrosine 701 but not phosphorylation of serine 727. The coactivator cyclic AMP response element-binding protein-binding protein (CBP) is an important component of induction of MMP-9 gene transcription. IFN-γ induces the in vivo association of STAT-1α and CBP and decreases the association of CBP to the MMP-9 promoter. IFN-γ does not influence the stability of CBP nor does IFN-γ affect chromatin-remodeling events on the MMP-9 promoter. IFN-γ inhibits the assembly of the MMP-9 transcription complex by suppressing H3/H4 acetylation and inhibiting recruitment of Pol II to the MMP-9 promoter. These findings indicate that IFN-γ/ STAT-1α exert their inhibitory effects by affecting multiple aspects of MMP-9 gene transcription. © Society for Leukocyte Biology.