There have been reports showing a protective role of nonsteroidal anti-inflammatory drugs (NSAIDs) against gastrointestinal cancers. CpG island hyper methylation (CIHM) of tumor suppressor genes is a major event in carcinogenesis. We investigated the CIHM status of non-cancerous gastric mucosa in chronic NSAID users and non-users and assessed the effect of NSAIDs on CIHM. Gastric mucosa samples were obtained from 51 chronic NSAID users and 180 non-users. CIHM of p14(ARF), p16(INK4a), death-associated protein kinase (DAP-kinase), and E-cadherin (CDH1) genes were determined by methylation-specific PCR. CIHM high was defined as two or more CpG islands methylated. CIHM of p14, p16, CDH1, and CIHM high were lower in chronic NSAID users than in non-users (p14: non-users vs users = 32.2%vs 9.8%, P = 0.003; p16: non-users vs users = 35.0%vs 15.7%, P = 0.02; CDH1: non-users vs users = 36.1%vs 9.8%, P = 0.0009; CIHM high: non-users vs users = 44.4%vs 17.6%, P = 0.0009). NSAID use was also associated with decreased number of CIHM by anova (R = -0.32, P < 0.0001). Multivariate logistic regression analysis with adjustment for sex, age, Helicobacter pylori infection, and NSAID use revealed that NSAID use was inversely correlated with all four CIHM and CIHM high as an independent factor (p14: odds ratio [OR] = 0.17, 95% confidence interval [CI] = 0.06-0.48; p16: OR = 0.32, 95% CI = 0.14-0.75; DAP-kinase: OR = 0.45, 95% CI = 0.22-0.92; CDH1: OR = 0.18, 95% CI = 0.06-0.48; CIHM high: OR = 0.21, 95% CI = 0.09-0.49). No association was found between CIHM status and the duration or dose of NSAIDs. Chronic NSAID use suppresses CIHM in human gastric mucosa. NSAIDs may have a suppressive role against methylation-related gastric carcinogenesis.