Hepatocyte growth factor (HGF) activator inhibitor type 2 (HAI-2/SPINT2) encodes Kunitz-type protease inhibitor that regulates HGF activity. Inspection of the human HAI-2/SPINT2 locus uncovered a large and dense CpG island within the 5' region of this gene. Analysis of cultured human gastric tumor lines indicated that HAI-2/SPINT2 expression is either undetectable or in low abundance in several lines; however, enhanced gene expression was measured in cells cultured on the DNA demethylating agent 5-aza-2'-deoxycytidine. Bisulfite DNA sequencing confirmed the densely methylated HAI-2/SPINT2 promoter region. Forced expression of HAI-2/SPINT2 induced cell apoptosis, suppressed anchorage independent growth in vitro and tumor growth in vivo. We investigated HAI-2/SPINT2 aberrant methylation in patients with gastric cancer. The HAI-2/SPINT2 methylation was found preferentially in cancerous tissues (30 of 40, 75%) compared with nontumor tissues (no methylation was detected), indicating that this aberrant characteristic is common in gastric malignancies. In conclusion, epigenetic inactivation of HAI-2/SPINT2 is a common event contributing to gastric carcinogenesis and may be a potential biomarker for gastric cancer.