Mitochondrial quality control is essential in modulating intramitochondrial ROS (mtROS) homeostasis that is a critical determinant for many human disorders. Here we identified an atypical function of a heat shock factor, Hsf3, as an important mtROS regulator. Despite HSFs are nuclear proteins and master regulators of protein unfolded response (UPR) across taxa, we demonstrate that Hsf3 is a both nuclei and mitochondria targeting transcription factor that plays an irrelevant function in controlling UPR process. Instead, Hsf3 represses TCA cycle genes and simultaneously regulates electron transfer chain genes encoded from both nuclei and mitochondria. Moreover, both nuclear and mitochondrial targeting signal are required for promising Hsf3 function. Hsf3 regulation responds to multiple intramitochondrial stresses, which activates and ameliorates Hsf3 DNA binding via oxidation of the cysteine residue on DNA binding domain of Hsf3. Collectively, our findings reveal a previously unknown role of HSF family in modulation of mitochondrial function and damage.