Elevated fumarate concentrations resulting from inhibition of the Krebs cycle lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH reoxidation can block Krebs cycle activity, therefore we hypothesized that oxidative phosphorylation deficiencies such as those observed in mitochondrial diseases would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly the vestibular nucleus (VN). Notably, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to VN pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS samples allowed us to identify the voltage-dependent anion channels (VDAC) 1 and 2 as specific targets of succination. Using targeted mass spectrometry, Cys77 was identified as a site of endogenous succination in VDAC2. Given the important role of VDAC isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial brain function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.