Mitochondrial reactive oxygen species (ROS) are believed to stabilize hypoxia-inducible factor (HIF)-1α, a transcriptional regulator of the immune response. Mclk1 encodes a mitochondrial protein that is necessary for ubiquinone biosynthesis. Heterozygote Mclk1+/− mutant mice are long-lived despite increased mitochondrial ROS and decreased energy metabolism. In this study, Mclk1+/− mutant mice in the C57BL/6J background displayed increased basal and induced expression of HIF-1α in liver and macrophages in association with elevated expression of inflammatory cytokines, in particular TNF-α. Mutant macrophages showed increased classical and decreased alternative activation, and mutant mice were hypersensitive to LPS. Consistent with these observations in vivo, knock-down of Mclk1 in murine RAW264. 7 macrophage-like cells induced increased mitochondrial ROS as well as elevated expression of HIF-1α and secretion of TNF-α. We used an antioxidant peptide targeted to mitochondria to show that altered ROS metabolism is necessary for the enhanced expression of HIF-1α, which, in turn, is necessary for increased TNF-α secretion. These findings provide in vivo evidence for the action of mitochondrial ROS on HIF-1α activity and demonstrate that changes in mitochondrial function within physiologically tolerable limits modulate the immune response. Our results further suggest that altered immune function through a limited increase in HIF-1α expression can positively impact animal longevity.