Background/Aims: Gastric cancer (GC) remains the second leading cause of cancer-related deaths in the world. Successful early cancer detection is hampered by lack of highly sensitive and specific biomarkers. Mitochondrial dysfunction contributes to an aggressive carcinogenic phenotype of many cancers. We hypothesized that changes in the mitochondrial proteome are required to support development of GC. Methods: TMT method followed by mass spectrometry analysis was utilized to quantify alterations in protein abundance in mitochondria enriched between noncancer and gastric cancer tissues. Results: Of a total data set that included 738 identified proteins, about 40.1% were found to be mitochondrial and associated proteins. Among them, 234 proteins were at least 1.5-fold up- or down-regulated in the gastric cancer compared with the adherent normal tissues. A number of markers (e.g. HSP70, HSP60, HSP90, leucine-rich pentatricopeptide repeat containing (LRPPRC), SOD2 and cathepsin B) were previously reported as biomarkers of GC. Additionally, several potential biomarkers participated in mitochondrial oxidative phosphorylation and active fatty acid oxidation were firstly identified differentially expressed in GC samples. Our findings also suggest that VDAC1 may be a novel biomarker for GC. Conclusion: The results show that subcellular proteomics of tumor tissue is feasible and a promising avenue for exploring oncogenesis.