DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a 465-kDa catalytic subunit of DNA-PK, a DNA repair apparatus. DNA-PKcs has been reported to be a tumor suppressor, but details of its expression in human cancer are controversial. To determine the protein expression and clinical implications of DNA-PKcs in gastric carcinogenesis and cancer progression, we evaluated its expression status by immunohistochemistry in 122 non-neoplastic gastric mucosa samples, and in 115 gastric adenomas and 564 consecutive gastric cancers. In addition, we evaluated the clinicopathologic characteristics of gastric cancers showing altered DNA-PKcs expression, and performed microsatellite instability (MSI) analysis at BAT-26 and frameshift mutation analysis of DNA-PKcs. DNA-PKcs expression was negative in foveolar epithelium of normal gastric mucosal tissues, but was positive in most Helicobacter pylori-associated gastritis, intestinal metaplasia and gastric adenoma tissues. In gastric cancers, negative expression of DNA-PKcs was found in 114 of the 564 (20.2%) cancers and was significantly associated with intratumoral neutrophils, MSI-high (H) phenotype, tumor progression, and poor patient survival (p<0.05). Frameshift mutations of (A)10 mononucleotide repeats in DNA-PKcs were found in 24.3% of MSI-H gastric cancers and these were associated with negative expression of DNA-PKcs. Although patients with MSI-H gastric cancers were found to have a lower risk of lymph node metastasis, gastric cancers harboring the (A)10 mutation of DNA-PKcs were found to have a higher risk of lymph node metastasis. In conclusion, the expression of DNA-PKcs was found to be altered during gastric carcinogenesis and negative DNA-PKcs expression was associated with gastric cancer progression. The (A)10 frameshift mutation of DNA-PKcs in gastric cancers was a target of defective mismatch repair, and was associated with lymph node metastasis.