ABSTRACT Introduction: Hepatitis B virus (HBV) is responsible for one of the most common human viral infections. An estimated 257 million people are living with chronic HBV infection worldwide, and mortality has reached 900,000 deaths in recent years. In 2001, the World Health Organization reported a prevalence of chronic hepatitis B infection in Iran between 2-7%. Objective: To assess the effect of the national HBV mass vaccination program after 25 years. Methods: A retrospective cohort study was conducted in vaccinated and unvaccinated people according to the year of birth. Blood samples were obtained from each enrolled person and data about demographic variables, and medical and vaccination history were collected using a standardized questionnaire. Persons were considered uninfected if they were negative for both HBsAg and anti-HBc. Also, Vaccine effectiveness was measured by calculating the risk of disease among vaccinated and unvaccinated persons and defining the percentage risk reduction of infection in the vaccinated group. Results: A total of 2720 persons were interviewed. The rate of HBV breakthrough infection among the vaccinated group was significantly lower than in unvaccinated group. One hundred ninety-four cases with positive HBV markers of infection were identified. The risk ratio of HBV infection was 0.71, 95% CI: 0.54-0.94 (vaccinated/unvaccinated). The estimated vaccination effectiveness against Hepatitis B infection was 29% (95% CI: 6%-46%). Conclusions: Iran has successfully combined hepatitis B vaccination into regular immunization programs. The WHO goal of reducing HBsAg prevalence to an equivalent of 1% by 2020 has been reached. With respect to vaccination effectiveness and low prevalence of the disease in the country, catch-up hepatitis B vaccination programs for adolescents can guarantee the immunity of the population.