Aim Although the prevalence of Helicobacter pylori (H.pylori) increases with age and the main period of acquisition is childhood, the route of transmission of H.pylori infection remains unclear. This study aims to evaluate the relationship between prevalence of children and grandparents. Methods A total of 838 consecutive children who attended the Urita clinic and whose blood was taken for work up were enrolled in the present study. They were 449 boys and 389 girls, with a mean age of 12.4 years. H.pylori serology of their family members who were living together in one house was picked up to analyse intra-familial clustering of H.pylori infection. The family members of these children consisted of 448 fathers, 597 mothers, 205 grandfathers, 361 grandmothers and 589 siblings. Results The seropositive rates of mothers, grandmother and siblings in seropositive children were significantly higher than those in seronegative children. H.pylori infection in mothers and grandmothers was a marked risk factor for infection in the index children. Larger family size was not a risk factor for H.pylori infection. In contrast, having an infected father or grandfather was not an independent predictor for children infection. Conclusions Our data demonstrate that not only mother-to-child transmission but also grandmother-to-child transmission is an important mechanism for the spread of H.pylori in a three-generation household.