OBJECTIVES: Helicobacter pylori (H. pylori) infection is a widely recognized factor in causing inflammation in the stomach and significantly increases the likelihood of developing gastric cancer. The relation between intake of vitamin B2 in the diet and testing positive for Helicobacter pylori is still unclear. METHODS: This cross-sectional study used data from the NHANES conducted in the United States in 1999-2000. Vitamin B2 consumption was measured from one 24-hour recall interview. H. pylori seropositivity was confirmed through an ELISA test measuring serum lgG protein levels. Multivariable logistic regression models, subgroup analysis and sensitivity analysis were performed to evaluate the possible association between dietary vitamin B2 consumption and H. pylori seropositivity. RESULTS: Out of the total 2,859 participants, 1,257 had H. pylori seropositivity, with males making up 47.5% and the mean age was 49.7 years old. There was a significant inverse association between vitamin B2 intake and H. pylori [odds ratio (OR): 0.88, 95% confidence interval (CI) (0.78-0.99), p = 0.031], after full adjustment for covariates. When dietary vitamin B2 analyzed as a categorical variable, the risk of H. pylori seropositivity in the highest quartile Q4 group decreased by 39% compared to lowest quartile Q1 (OR 0.61, 95% CI 0.44-0.86, p = 0.004, Model 3). The findings of subgroup analysis and sensitivity analysis were constant and dependable. CONCLUSION: This study implies that increased dietary vitamin B2 consumption may be related with a decreased incidence of Helicobacter pylori seropositivity.