Macrophage activation by the proinflammatory cytokine interferon-γ (IFN-γ) is a critical component of the host innate response to bacterial pathogenesis. However, the precise nature of the IFN-γ-induced activation pathway is not known. Here we show using genome-wide expression and chromatin-binding profiling that IFN-γ induces the expression of many nuclear genes encoding mitochondrial respiratory chain machinery via activation of the nuclear receptor ERRα (estrogen-related receptor α, NR3B1). Studies with macrophages lacking ERRα demonstrate that it is required for induction of mitochondrial reactive oxygen species (ROS) production and efficient clearance of Listeria monocytogenes (LM) in response to IFN-γ. As a result, mice lacking ERRα are susceptible to LM infection, a phenotype that is localized to bone marrow-derived cells. Furthermore, we found that IFN-γ-induced activation of ERRα depends on coactivator PGC-1β (peroxisome proliferator-activated receptor γ coactivator-1β), which appears to be a direct target for the IFN-γ/STAT-1 signaling cascade. Thus, ERRα and PGC-1β act together as a key effector of IFN-γ-induced mitochondrial ROS production and host defense.