Mitochondrial response to inflammation is crucial in the metabolic adaptation to infection. This study aimed to explore the mitochondrial response under inflammatory and anti-inflammatory environments, with a focus on the tricarboxylic acid (TCA) cycle. Expression levels of key TCA cycle enzymes and the autophagy-related protein light chain 3b (LC3b) were determined in raw 264.7 cells treated with lipopolysaccharide (LPS) and metformin (Met). Additionally, reactive oxygen species (ROS) levels and mitochondrial membrane potential were assessed using flow cytometry. Moreover, 8-week-old C57BL/6J mice were intraperitoneally injected with LPS and Met to assess the mitochondrial response in vivo. Upon LPS stimulation, the expression of key TCA enzymes, including citrate synthase, α-ketoglutarate dehydrogenase, and isocitrate dehydrogenase 2, and the mitochondrial membrane potential decreased, whereas the levels of LC3b and ROS increased. However, treatment with Met inhibited the reduction of LPS-induced enzyme levels as well as the elevation of LC3b and ROS levels. In conclusion, the mitochondrial TCA cycle is affected by the inflammatory environment, and the LPS-induced effects can be reversed by Met treatment.