Isolated methylmalonic acidemia (MMA) is a rare metabolic disease due to the deficient activity of L-methylmalonyl-CoA mutase (MCM). This mitochondrial enzyme converts L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (Adocbl) as cofactor. Isolated MMA is subdivided into five forms: mut MMA associated with MCM deficiency, three different defects related to mitochondrial Adocbl formation (cblA, cblB, and cblH), and cblD variant 2. We performed proteomic analysis on mitochondria from an individual with cblH/cblD disorder using 2-D DIGE to identify differentially expressed proteins in this disease. Comparative analysis of control/patient mitochondrial proteome allowed us to identify differential expression of 10 proteins. The most notable groups included proteins involved in apoptosis (cytochrome c), oxidative stress (manganese superoxide dismutase) and cell metabolism (succinyl-CoA ligase (GDP forming) and mitochondrial glycerophosphate dehydrogenase). Immunoblot analysis further validated 2-D DIGE results of two of these proteins in multiple MMA patients, suggesting that the differences in expression are a general effect in this disorder. It is feasible that the differential proteins identified in this study have a biological significance and might be related to the pathophysiology of MMA.