Background: Phosphorylation modification, as one kind of protein post-translational modification (PTM), increases the diverse protein functions, such as cell signaling, protein folding, enzymatic activity, protein degradation, and their subcellular location. However, the subcellular phophoproteomic of ovarian carcinoma mitochondrial proteins has not been elucidated. Methods: Here, an 8-plex isobaric tag for relative and absolute quantification (iTRAQ) proteomics was used to identify mitochondrial expressed proteins (mtEPs) and phosphorylation modification sites of ovarian carcinomas relative to controls, followed by bioinformatics analysis. Results: The iTRAQ-based proteomics identified 5115 proteins and 99 phosphorylated proteins with quantitative information from purified mitochondrial samples, and 262 proteins were significantly related to overall survival in ovarian cancer patients. Interestingly, the results demonstrated that cancer cells exhibit an increased dependence on lipid metabolism, such as biosynthesis of unsaturated fatty acids, butanoate metabolism, fatty acid degradation, fatty acid metabolism, which might play an important role in ovarian carcinoma invasion and metastasis. Moreover 33 proteins related to lipid metabolism as potential markers for the development of ovarian carcinoma were identified.Additionally, 3 drug-associated phosphorylation proteins and 3 phosphorylation proteins as tumor markers in the plasma were obtained. The 99 phosphorylated proteins and TCGA data were integarated, thus obtained 9 important proteins. Among those, HSP60 were highly related with lipid metabolism. In cells with increased Hsp60 levels both the amounts of total mitochondrial short-chain acyl-CoA dehydrogenase (ACADS) proteins and folded ACADS were increased, which may influence mitochondrial protein folding and lipid metabolism. Conclusions: The current study provides a large-scale mitochondrial proteomic profiling and mitochondrion phosphoproteome with quantitative information, a certain number of proteins with the potential biomarkers, drug targets and a novel vision in the lipid metabolism bio-mechanism of human ovarian carcinoma. Legal entity responsible for the study: Hunan Engineering Laboratory for Structural Biology and Drug Design, Xiangya Hospital, Central South University. Funding: Xiangya Hospital Funds for Talent Introduction (to XZ), the grants from China “863” Plan Project (Grant No. 2014AA020610-1 to XZ), the National Natural Science Foundation of China (Grant No. 81272798 and 81572278 to XZ), and the Hunan Provincial Natural Science Foundation of China (Grant No. 14JJ7008 to XZ). Disclosure: All authors have declared no conflicts of interest.