Mitochondria exhibit diverse effects on cellular responses. Mitochondrial transplantation from gastric epithelial cells GES-1 to gastric cancer cells AGS reduces cancer malignancy was previously reported. We employed proteomic and metabolomic analyses to elucidate underlying mechanisms. iTRAQ/TMT-based proteomics identified 257 upregulated and 34 downregulated proteins, with Ingenuity pathway analysis revealing regulation of 14 signaling pathways. The upregulation of p53, Bax, p-AktS473, p-mTORS2448 and the down-regulation of Sirt 3, p-NRF2S40, and HO-1 were further verified by western blotting. Metabolomic profiling detected 3 upregulated and 8 down-regulated metabolites implicated in glycolysis, TCA cycle, pentose phosphate pathway (PPP), and ATP production. Further investigation showed that decreased extracellular lactate correlating with enhanced MCT1 expression (lactate importer), reduced MCT4 expression (lactate exporter), and increased LDHB expression (lactate to-pyruvate conversion). With the finding that transplanted with GES-1 mitochondria and induced accumulation of pyruvate, the AGS was solely treated with pyruvate and the result showed a cell migration of AGS was retarded. Additionally, isocitrate accumulation preceded decreased α-ketoglutarate, malate, ATP, and NADH levels. In conclusion, integrated proteomic and metabolomic analyses revealed that transplanted GES-1 mitochondria attenuate AGS gastric cancer malignancy through stagnation of glycolysis and the TCA cycle progression, highlighting potential targets for mitochondrial-based therapies.