Sepsis causes many early deaths; both macrophage mitochondrial damage and oxidative stress responses are key factors in its pathogenesis. Although the exact mechanisms responsible for sepsis-induced mitochondrial damage are unknown, the nuclear transcription factor, interferon regulatory factor-1 (IRF-1) has been reported to cause mitochondrial damage in several diseases. Previously, we reported that in addition to promoting systemic inflammation, IRF-1 promoted the apoptosis of and inhibited autophagy in macrophages. In the present study, we hypothesized that lipopolysaccharide (LPS)-induced IRF-1 activation in macrophages may promote mitochondrial damage and oxidative stress. In vitro, LPS was found to promote IRF-1 activation, reactive oxygen species (ROS) production, adenosine triphosphate (ATP) depletion, superoxide dismutase (SOD) consumption, malondialdehyde (MDA) accumulation and mitochondrial depolarization in macrophages in a time- and dose-dependent manner. These effects were abrogated in cells in which IRF-1 was knocked down. Furthermore, IRF-1 overexpression increased LPS-induced oxidative stress responses and mitochondrial damage. In vivo, peritoneal macrophages obtained from IRF-1 knockout (KO) mice produced less ROS and had less mitochondrial depolarization and damage following the administration of LPS, when compared to their wild-type (WT) counterparts. In addition, IRF-1 KO mice exhibited a decreased release of mitochondrial DNA (mtDNA) following the administration of LPS. Thus, IRF-1 may be a critical factor in augmenting LPS-induced oxidative stress and mitochondrial damage in macrophages.