BACKGROUND Lymph node metastasis (LNM) is recognized as an important factor in the progression of tumor malignancy. It is required to discover molecular markers for the prediction of LNM in gastric cancers (GCs). METHODS An isotope coded affinity tag (ICAT) method and mass spectrometry were used for the quantitative profiling of LNM-related proteins. Western blot analysis of the identified proteins and immunohistochemistry on a tissue microarray comprising 120 GC cases were performed for validation. RESULTS We identified 151 differentially expressed proteins (DEPs) with an abundance ratio greater than 1.5-fold. The proteins upregulated in LNM-negative GCs were largely populated with proteins related to cell death. Among the DEPs, galectin-2 was further tested because its expression level was significantly higher in LNM-negative GCs (~12-fold, p < 0.0001) and its expression is known to be not ubiquitous but confined to the gastrointestinal tract. Immunohistochemical analysis revealed that low expression of galectin-2 was significantly associated with LNM (p = 0.031) and advanced clinical stage (p = 0.024). The association of low galectin-2 with LNM was found even in early GCs (p = 0.020). CONCLUSIONS Our results show that proteomic analysis coupled with immunohistochemistry using tissue microarray is a useful tool for identifying LNM-associated proteins in GC. Also, loss of galectin-2 might play an important role in the aggressiveness of GC.