The levels of organic acids representing metabolic pathway end products are important indicators of physiological status, and may be associated with metabolic changes in cancer. The aim of this study is to investigate the levels of organic acids in cancerous and normal tissues from gastric cancer patients and to confirm the role of metabolic alterations in gastric carcinogenesis. Organic acids in normal and cancerous tissues from forty-five patients with gastric adenocarcinoma were investigated by gas chromatography-mass spectrometry in selected ion monitoring mode as methoxime/tert-butyldimethylsilyl derivatives. We analysed the significant differences in the levels of organic acids in normal and cancer tissues and investigated the correlation of these levels in cancer tissues with clinicopathological features. The levels of Krebs cycle components, including α-ketoglutaric acid, succinic acid, fumaric acid, malic acid and oxaloacetic acid, were significantly increased in cancer tissues compared to normal tissues. In addition, the levels of glycolytic products, including pyruvic acid and lactic acid, as well as the levels of ketone bodies, including 3-hydroxybutyric acid, were also significantly increased in cancer tissues compared to normal tissues. The levels of ketone bodies in cancer tissues with differentiated histology and in intestinal-type cancer tissues were significantly increased. The organic acid profiling analysis described here may be a generally useful clinical tool for understanding the complexity of metabolic events in gastric adenocarcinoma, and organic acids may have potential as metabolic markers for the future discovery of diagnostic and therapeutic modalities.