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Itaconate is a dicarboxylic acid that inhibits the isocitrate lyase enzyme of the bacterial glyoxylate shunt. Activated macrophages have been shown to produce itaconate, suggesting that these immune cells may employ this metabolite as a weapon against invading bacteria. Here, we demonstrate that in vitro, itaconate can exhibit bactericidal effects under acidic conditions similar to the pH of a macrophage phagosome. In parallel, successful pathogens, including Salmonella, have acquired a genetic operon encoding itaconate degradation proteins, which are induced heavily in macrophages. We characterized the regulation of this operon by the neighboring gene ripR in specific response to itaconate. Moreover, we developed an itaconate biosensor based on the operon promoter that can detect itaconate in a semiquantitative manner and, when combined with the ripR gene, is sufficient for itaconate-regulated expression in Escherichia coli Using this biosensor with fluorescence microscopy, we observed bacteria responding to itaconate in the phagosomes of macrophages and provide additional evidence that gamma interferon stimulates macrophage itaconate synthesis and that J774 mouse macrophages produce substantially more itaconate than the human THP-1 monocyte cell line. In summary, we examined the role of itaconate as an antibacterial metabolite in mouse and human macrophages, characterized the regulation of Salmonella's defense against it, and developed it as a convenient itaconate biosensor and inducible promoter system.