Helicobacter pylori infection is the most important risk factor for gastric cancer. It is acquired predominantly during childhood, and understanding the determinants of infection in early life may translate into identifying preventive measures. However, the independent role of child day-care attendance remains to be understood. The aim of the study was to evaluate the association between child day-care attendance and H. pylori infection in early life. The study was nested within Geração XXI, a birth cohort assembled in Portugal. Serum anti-H. pylori IgG was quantified using ELISA in 1047 children between the ages of 4 and 5 years, and information on child day-care attendance since birth was collected. Odds ratios and 95% confidence intervals (95% CIs), adjusted for the child’s age and number of siblings, as well as maternal education and infection status, were computed using unconditional logistic regression. The prevalence of H. pylori infection was 30.6% (95% CI 27.9–33.6), and it increased significantly with the cumulative time of attendance in day-care centers/homes (from 13.2% among never attendees to 40.2% among those attending for >36 months; P for trend<0.001). The odds ratio was 4.88 (95% CI 2.55–9.35) among those attending these institutions for more than 3 years, in comparison with never attendees. H. pylori infection remains a frequent and early event in Portugal. Child day-care attendance increases the risk of infection, making this setting a target for preventive measures.