To evaluate the association between RUNX3 expression and Helicobacter pylori (H. pylori)-associated precancerous gastric lesions. A population-based study was conducted in Linqu County, Shandong Province, a high-risk area of gastric cancer in China. RUNX3 expression was determined by immunohistochemistry analysis in 1 026 H. pylori infected subjects with different gastric lesions. Among 1,026 subjects, 359 (35.0%, 359/1,026) was positive. The prevalence rates of RUNX3 expression decreased steadily with severity of gastric lesions, 65.6%(40/61) among those with superficial gastritis /normal (SG/N), and 22.4%(60/268) among those with dysplasia (DYS) (P<0.01). Multinomial logistic regression analysis indicated that the risk of advanced gastric lesions were significantly increased for chronic atrophic gastritis [CAG, odds ratio (OR)=2.83, 95% confidence interval (CI: 1.58 to 5.06), intestinal metaplasia (IM, OR=3.52, 95% CI: 1.98 to 6.24) and DYS (OR=7.19, 95% CI: 3.81 to 13.56)] in subjects with negative RUNX3 expression compared with positive. Furthermore, compared to subjects with positive RUNX3 expression and non-smoking (or non-drinking), significantly increased risk of IM or DYS was observed in subjects with negative RUNX3 expression, the ORs ranged from 2.78 to 12.29. Our findings indicate a significantly negative correlation between RUNX3 expression and severity of H. pylori-associated gastric lesions, suggesting that decreased expression of RUNX3 may play an important role in evolution of precancerous gastric lesions.