The declining incidence of hepatitis A virus (HAV) infection in the Islamic Republic of Iran may be reducing the population’s natural immunity. This was the first systemized, population-based survey of the seroprevalence of HAV antibodies in urban and rural inhabitants of Sari, Mazandaran province. Serum from 1034 individuals aged 1–25 years in 2007 were tested for anti-HAV IgG antibody using a commercial enzyme immunoassay kit. The overall seroprevalence was 38.9%. The lowest prevalence (5.2%) was among the younger age group (1–5 years) from urban areas and the highest prevalence (82.0%) in the older age group (15–25 years) from rural areas. Seropositivity was significantly higher at higher age, among females and in rural areas. Sari is no longer classified as an area of high endemicity, and immunization against HAV may be needed in our population in the future.