Mycobacteria survive in macrophages despite triggering pattern recognition receptors and T cell-derived IFN-g production. Mycobacterial cord factor trehalose-6,6-dimycolate (TDM) binds the C-type lectin receptor MINCLE and induces inflammatory gene expression. However, the impact of TDM on IFN-g-induced macrophage activation is not known. In this study, we have investigated the cross-regulation of the mouse macrophage transcriptome by IFN-g and by TDM or its synthetic analogue trehalose-6,6-dibehenate (TDB). As expected, IFN-g induced genes involved in Ag presentation and antimicrobial defense. Transcriptional programs induced by TDM and TDB were highly similar but clearly distinct from the response to IFN-g. The glycolipids enhanced expression of a subset of IFN-g-induced genes associated with inflammation. In contrast, TDM/TDB exerted delayed inhibition of IFN-g-induced genes, including pattern recognition receptors, MHC class II genes, and IFN-g-induced GTPases, with antimicrobial function. TDM downregulated MHC class II cell surface expression and impaired T cell activation by peptide-pulsed macrophages. Inhibition of the IFN-g-induced GTPase GBP1 occurred at the level of transcription by a partially MINCLE-dependent mechanism that may target IRF1 activity. Although activation of STAT1 was unaltered, deletion of Socs1 relieved inhibition of GBP1 expression by TDM. Nonnuclear Socs1 was sufficient for inhibition, suggesting a noncanonical, cytoplasmic mechanism. Taken together, unbiased analysis of transcriptional reprogramming revealed a significant degree of negative regulation of IFN-g-induced Ag presentation and antimicrobial gene expression by the mycobacterial cord factor that may contribute to mycobacterial persistence. Copyright © 2020 by The American Association of Immunologists, Inc.