Alterations in energy metabolism play a major role in cancer development. Aconitase (ACO2) is an essential enzyme located in the mitochondria and catalyzes the interconversion of citrate and isocitrate in the tricarboxylic acid cycle. Recent studies suggest that the expression of ACO2 may be altered in certain types of cancer. The purpose of this study was to examine ACO2 expression in clinical tumor specimens from patients with gastric cancer and to evaluate the clinical relevance of ACO2 expression in gastric cancer. A total of 456 paraffin-embedded gastric cancer tissues and 30 pairs of freshly frozen tissues were used in this study. Real-time quantitative reverse transcription polymerase chain reaction, western blotting, and immunohistochemical staining were performed to measure ACO2 expression in tumor tissues and matched adjacent non-tumorous tissues. The results showed that the expression of ACO2 was significantly down-regulated in gastric cancer tissues compared with matched adjacent non-tumorous tissues and was associated with clinical stage (p = 0.001), T classification (p = 0.027), N classification (p = 0.012), M classification (p = 0.002), and pathological differentiation states (p = 0.036). Patients with lower ACO2 expression had a shorter survival time than those with higher ACO2 expression. Univariate and multivariate analyses indicated that ACO2 expression functions as an independent prognostic factor (p < 0.001). Our data suggested that ACO2 could play an important role in gastric cancer and may potentially serve as a prognostic biomarker.