Runx3, a member of the human runt-related transcription factor family, is known as a possible tumor suppressor gene for gastric cancer. Runx 3 expression is frequently suppressed by the promoter hypermethylation in gastric cancer cell lines and tissues. However, the precise mechanism of the induction of Runx3 methylation, which is considered to be a critical step in gastric carcinogenesis, remains to be elucidated. In the present study, we evaluated runx3 gene methylation in 57 resected early gastric cancer specimens. Then, we correlated Runx3 methylation in the cancer tissue specimens with clinicopathological factors as well as the mucosal backgrounds, such as intestinal metaplasia surrounding the cancer cells and Helicobacter pylori (H. pylori) infection. Runx3 methylation was observed in 30 of the 57 (52.6%) cancer specimens, whereas methylation was detected in 10 of the 57 (17.5%) corresponding non-cancerous mucosae. In comparison to the clinicopathological factors, Runx3 methylation was significantly correlated with both age and tumor location. A multivariate analysis demonstrated that age and tumor location as well as H. pylori infection were independent risk factors for Runx3 methylation. We demonstrated for the first time that H. pylori infection contributes to Runx3 methylation in gastric cancer tissues. When a persistent infection by H. pylori continues in the middle/lower stomach for a long period, Runx3 methylation may be induced and the subsequent loss of Runx3 expression may therefore affect gastric carcinogenesis.