AbstractGastric cancer (GC) is one of the most malignant tumors worldwide. Despite the recent decrease in mortality rates, the prognosis remains poor. Therefore, it is necessary to find novel biomarkers with early diagnostic value for GC. In this study, we present a large-scale proteomic analysis of 30 GC tissues and 30 matched healthy tissues using label-free global proteome profiling. Our results identified 537 differentially expressed proteins, including 280 upregulated and 257 downregulated proteins. The ingenuity pathway analysis (IPA) results indicated that the sirtuin signaling pathway was the most activated pathway in GC tissues whereas oxidative phosphorylation was the most inhibited. Moreover, the most activated molecular function was cellular movement, including tissue invasion by tumor cell lines. Based on IPA results, 15 hub proteins were screened. Using the receiver operating characteristic curve, most of hub proteins showed a high diagnostic power in distinguishing between tumors and healthy controls. A four-protein (ATP5B-ATP5O-NDUFB4-NDUFB8) diagnostic signature was built using a random forest model. The area under the curve (AUC) of this model was 0.996 and 0.886 for the training and testing set, respectively, suggesting that the four-protein signature has a high diagnostic power. This signature was further tested with independent datasets using plasma enzyme-linked immune sorbent assays, resulted in an AUC of 0.778 for distinguishing GC tissues from healthy controls, and using immunohistochemical tissue microarray analysis, resulting in an AUC of 0.805. In conclusion, this study identifies potential biomarkers and improves our understanding of the pathogenesis, providing novel therapeutic targets for GC.