Background: Helicobacter pylori is a common gastrointestinal pathogen in human. The mechanism of its acquisition and transmission is unclear, although the most likely mode of transmission is fecal-oral or oral-oral. Most of infections occur in childhood. The current cohort assessed the prevalence of infection in infants with different feeding strategies (exclusive breastfeeding, formula, or mixed feeding). Methods: The current study was carried out in Vali-Asr hospital of Imam Khomeini hospital complex, Tehran, Iran, from 2012 to 2015.The study population included infants born in this hospital and registered in breastfeeding research center during the first 6 months of life. They were compared in 2 groups; exclusively breastfed, and formula or mixed fed. Mothers' H. pylori infection was checked serologically using the enzyme-linked immunosorbent assay (ELISA) technique and those of infants by monoclonal antibody test using ELISA technique on stool samples at 1st and 6th month of life. Results: A total of 102 infants in 2 groups (54 exclusively breastfed and 48 formula or mixed fed) completed the study. Mothers' serological assessments were positive in 68% of exclusively breast-fed subjects and 60% of formula or mixed fed subjects. All 1-monthold infants were negative for the infection and in the 6-month-old infants 10 cases (18.5%) were positive, 1 case was at borderline in the exclusively breastfed, and 13 cases (27%) were positive in formula-or mixed-fed subjects. This difference was not statistically significant (P = 0.302). The Mantel-Hanzel test was used to find a relationship in H. pylori positivity in mothers and infants during the first 6-month of life in each of the 2 groups. It was found that out of 37 serologically positive mothers in the breastfed group, 8 infants (22%) were H. pylori positive and in the formula and mixed-fed group, of 29 serologically positive mothers 10 infants (34%) were also H. pylori positive, but the difference between the groups was not statistically significant (P = 0.727). It meant that the prevalence of infantile H. pylori infection was similar in the 2 groups. Conclusions: The current study findings showed that exclusive breastfeeding had no role in protection or facilitation of H. pylori infection.