IFN-γ is essential for resistance to many intracellular pathogens, including Mycobacterium tuberculosis. Transcription of the IFN-γ gene in activated T cells is controlled by the proximal promoter element (-73 to -48 bp). CREB binds to the IFN-γ proximal promoter, and binding is enhanced by phosphorylation of CREB. Studies in human T cell lines and in transgenic mice have yielded conflicting results about whether CREB is a positive or a negative regulator of IFN-γ transcription. To determine the role of CREB in mediating IFN-γ production in response to a microbial pathogen, we evaluated the peripheral blood T cell response to M. tuberculosis in healthy tuberculin reactors. EMSAs, chromatin immunoprecipitation, and Western blotting demonstrated that stimulation of PBMC with M. tuberculosis induced phosphorylation and enhanced binding of CREB to the IFN-γ proximal promoter. Neutralization of CREB with intracellular Abs or down-regulation of CREB levels with small interfering RNA decreased M. tuberculosis-induced production of IFN-γ and IFN-γ mRNA expression. In addition, M. tuberculosis-stimulated T cells from tuberculosis patients, who have ineffective immunity, showed diminished IFN-γ production, reduced amounts of CREB binding to the IFN-γ proximal promoter, and absence of phosphorylated CREB. These findings demonstrate that CREB positively regulates IFN-γ production by human T cells that respond to M. tuberculosis.