The mitochondrial unfolded protein response (UPRmt) is a transcriptional program aimed at restoring proteostasis in mitochondria. Upregulation of mitochondrialmatrix proteases and heat shock proteins was initially described. Soon thereafter, a distinct UPRmt induced bymisfolded proteins in themitochondrial intermembrane space (IMS) andmediated by the estrogen receptor alpha (ERα), was found to upregulate the proteasome and the IMS protease OMI. However, the IMS-UPRmt was never studied in a neurodegenerative disease in vivo. Thus, we investigated the IMS-UPRmt in the G93A-SOD1mousemodel of familial ALS, sincemutant SOD1 is known to accumulate in the IMS of neural tissue and cause mitochondrial dysfunction. As the ERα is most active in females, we postulated that a differential involvement of the IMS-UPRmt could be linked to the longer lifespan of females in the G93A-SOD1mouse. We found a significant sex difference in the IMS-UPRmt, because the spinal cords of female, but notmale, G93A-SOD1mice showed elevation of OMI and proteasome activity. Then, using a mouse in which G93A-SOD1 was selectively targeted to the IMS, we demonstrated that the IMS-UPRmt could be specifically initiated bymutant SOD1 localized in the IMS. Furthermore, we showed that, in the absence of ERα, G93A-SOD1 failed to activate OMI and the proteasome, confirming the ERα dependence of the response. Taken together, these results demonstrate the IMS-UPRmt activation in SOD1 familial ALS, and suggest that sex differences in the disease phenotype could be linked to differential activation of the ERα axis of the IMS-UPRmt.