We recently demonstrated mitochondrial localization of estrogen receptor β (ERβ). We herein confirm the mitochondrial localization of ERβ by the loss of mitochondrial ERβ immunoreactivity in ERβ knockdown cells. Aphenotype change characterized as an increase in resistance to oxidative stressors is associated with ERβ knockdown. ERβ knockdown results in a lower resting mitochondrial membrane potential (Δψm) and increase in resistance to hydrogen peroxide-induced Δψm depolarization in both immortal hippocampal cells and primary hippocampal neurons. ERβ knockdown cells maintained ATP concentrations despite insults that compromise ATP production and produce less mitochondrial superoxide under oxidative stress. Furthermore, similar mitochondrial phenotype changes were identified in primary hippocampal neurons derived from ERβ knock-out mice. These data demonstrate that ERβ is expressed in mitochondria and function as a mitochondrial vulnerability factor involved in Δψm maintenance, potentially through a mitochondrial transcription dependent mechanism.