Signal Transducer and Activator of Transcription 3 (STAT3) has been tied to various physiological and pathological functions, mainly as a transcription factor that translocates to the nucleus upon tyrosine phosphorylation induced by cytokine stimulation. In addition, a small pool of STAT3 resides in the mitochondria where itserves as a sensor for various metabolic stressors including reactive oxygen species (ROS). Mitochondrially-localized STAT3 largely exerts its effects through direct or indirect regulation of the activity of the electron transport chain (ETC). It has been assumed that STAT3 amounts in the mitochondria are static. We showed that various stimuli, including oxidative stress and cytokines, triggered a signaling cascade thatresulted in a rapid loss of mitochondrially-localized STAT3. The loss of STAT3 from the mitochondria is due to its proteolysis. Recovery of the mitochondrial pool of STAT3 over time depended upon phosphorylation of Ser727 in STAT3 and new protein synthesis. Under these conditions, mitochondrially-localizedSTAT3 becomes competent to bind to cyclophilin D (CypD). Binding of STAT3 toCypD was mediated by the N-terminus of STAT3, which was also important for reducing mitochondrial ROS production after oxidative stress. These results outline a role formitochondrially-localized STAT3 in sensing and responding to external stimuli.