Gastric cancer is the second most common cancer worldwide and the fifth leading cause of cancer‐related death in Taiwan. Identification of biomarkers is essential to improve patient survival. Fifty aberrantly expressed proteins were identified using 2‐DE combined with MALDI TOF MS and were grouped based on their function. The overexpression of proteins was confirmed using real‐time quantitative RT‐PCR, Western blot, and immunohistochemical analysis. The clinicopathological correlations and prognostic significance of these aberrantly expressed proteins were evaluated to determine the novel gastric cancer biomarkers. In this study, expression of chloride intracellular channel 1 (CLIC1) is significantly up‐regulated in 67.9% of gastric patients and was selected for further study. The CLIC1 expression in tumor tissues was increased by 1.95‐fold (range, 0.01–6.19‐fold) compared with that expressed by adjacent noncancerous mucosa. Elevated CLIC1 expression was strongly correlated with lymph node metastasis, lymphatic invasion, perineural invasion, and pathological staging. Additionally, the 5‐year survival rate for the low CLIC1 expression group (n = 28; <1.72‐fold) was higher than that for the high CLIC1 expression group (n = 28; ≥1.72‐fold) (log rank, p = 0.0300). Experimental results indicate that overexpression of CLIC1 is a potential prognostic marker for gastric cancer.