Mycobacterium tuberculosis is an intracellular pathogen that persists within macrophages and remains a considerable global threat to human health. The purpose of this study was to investigate how interleukin (IL)-12 and IL-27 regulate human macrophage interactions with M. tuberculosis. Quantitative measurement of transcripts showed that IL-12 or M. tuberculosis induced IL-27 gene expression in human macrophages. Furthermore, IL-27 receptor subunits were shown by reverse transcription-polymerase chain reaction and flow cytometry to be expressed and present at the cell surface. Neutralization of IL-27 in the presence of IL-12 reduced viable M. tuberculosis recovered from macrophages. Antimycobacterial activity was accompanied by a heightened inflammatory response that included tumor necrosis factor, IL-6, interferon-gamma, and a subset of chemokines. These results implicate IL-12 and IL-27 in regulating human macrophages, and IL-27 derived from macrophages during infection impedes control of M. tuberculosis growth.