BACKGROUND: While Helicobacter pylori (H. pylori) is the major cause of gastric cancer, it has also been suggested to be involved in colorectal cancer (CRC) development. However, prospective studies addressing H. pylori and CRC are sparse and inconclusive. We assessed the association of antibody responses to H. pylori proteins with CRC in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. METHODS: We applied H. pylori multiplex serology to measure antibody responses to 13 H. pylori proteins in pre-diagnostic serum samples from 485 CRC cases and 485 matched controls nested within the EPIC study. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using multivariable conditional logistic regression to estimate the association of H. pylori overall and protein-specific sero-positivity with odds of developing CRC. RESULTS: Fifty-one percent of CRC cases were H. pylori sero-positive compared to 44% of controls resulting in an OR of 1.36 (95% CI: 1.00-1.85). Among the 13 individual H. pylori proteins, the association was driven mostly by sero-positivity to Helicobacter cysteine-rich protein C (HcpC) (OR: 1.66, 95% CI: 1.19-2.30) and Vacuolating cytotoxin A (VacA) (OR: 1.34, 95% CI: 0.99-1.82), the latter being non-statistically significant only in the fully adjusted model. CONCLUSION: In this prospective multi-center European study, antibody responses to H. pylori proteins, specifically HcpC and VacA, were associated with an increased risk of developing CRC. IMPACT: Biological mechanisms for a potential causal role of H. pylori in colorectal carcinogenesis need to be elucidated, and subsequently whether H. pylori eradication may decrease CRC incidence.