Infection with Helicobacter pylori is associated with significantly reduced risks of oesophageal adenocarcinoma; however, few studies have examined the association between H. pylori and Barrett's oesophagus (BO), the precursor lesion. We explored the relationship between H. pylori infection and BO and sought to identify potential modifiers. We compared the prevalence of positive H. pylori serology among 217 adults with simple BO (without dysplasia), 95 with dysplastic BO and 398 population controls sourced from the metropolitan Brisbane area. We determined H. pylori serostatus using enzyme-linked immunosorbent assay. To estimate relative risks, we calculated odds ratios (OR) and 95% confidence intervals (CI) using multivariable logistic regression in the entire sample and stratified by factors known to cause BO. The prevalence of H. pylori seropositivity was 12%, 3%, and 18%, respectively, among patients with simple BO, dysplastic BO and population controls. BO patients were significantly less likely to have antibodies for H. pylori (Simple BO: OR = 0.51, 95% CI: 0.30–0.86; Dysplastic BO: OR = 0.10, 95% CI: 0.03–0.33) than population controls. For simple BO, the association was diminished after adjustment for frequency of gastro-oesophageal reflux (GOR) symptoms. Adjustment for frequency of GOR symptoms did not substantially alter the observed effect for dysplastic BO. Although there was some variation in the magnitude of risk estimates across strata of age, sex, GOR symptoms and use of proton pump inhibitors or H2-receptor antagonists, the differences were uniformly nonsignificant. Helicobacter pylori infection is inversely associated with BO, and our findings suggest that decreased acid load is not the only mechanism underlying the H. pylori protective effect.