The fungal pathogen Histoplasma capsulatum evades the innate and adaptive immune responses and thrives within resting macrophages. Cytokines that induce antimicrobial activity, such as granulocyte macrophage colony-stimulating factor (GM-CSF), inhibit H. capsulatum growth in macrophages. Conversely, interleukin 4 inhibits the killing of intracellular pathogens. Using inductively coupled plasma mass spectrometry, we examined alterations in the metal homeostasis of murine H. capsulatum-infected macrophages that were exposed to activating cytokines. Decreases in the levels of iron (Fe²⁺ and Fe³⁺) and zinc (Zn²⁺) were observed in infected, GM-CSF-treated macrophages compared with those in infected controls. Interleukin 4 reversed the antifungal activity of GM-CSF-activated macrophages and was associated with increased intracellular Zn²⁺ levels. Chelation of Zn²⁺ inhibited yeast replication in both the absence of macrophages and the presence of macrophages. Treatment of cells with GM-CSF altered the host Zn²⁺ binding species profile. These results establish that Zn²⁺ deprivation may be a host defense mechanism utilized by macrophages.