Hepatitis A is a common infectious disease worldwide that was endemic in many regions of Southern Italy, such as Apulia region. After a large hepatitis A outbreak occurred between 1996 and 1997, in Apulia an active-free immunization program that was targeted to new-borns and adolescents was started. The aim of this study is to investigate the hepatitis A seroprevalence in the adult Apulian population 18 years after the immunization program introduction, in order to evaluate the risk of new epidemics onset.The study was carried out from May 2011 to June 2012 among blood donors from Department of Transfusion Medicine and Blood Bank of Policlinico General Hospital in Bari. Participants signed a written consent and filled out a questionnaire including items on demographic characteristics, risk factors, disease memory, and raw food consumption. Serum samples, collected from each patient, were tested for anti-HAV using the chemiluminescent microparticle immunoassay. Vaccination status against hepatitis A was checked on Regional Digital Immunization Registry (GIAVA).In total 1827 donors agreed to participate (77.7% male) with a mean age of 38.4 ± 11.7 years. However, 1172 (64.1%) donors were seropositive with no difference by sex. The highest proportion of seronegative subjects was in the 27 to 35 years age group. 91.8% of 1-dose vaccinated subjects (n = 190/207; 95%CI = 87.2-95.1) and 96.1% (n = 171/178; 95%CI = 92.1-98.1) of 2-doses vaccinated subjects were immune to the disease. Sensitivity of disease memory in unvaccinated subjects was 14.4% (95%CI = 12.2-16.7), specificity was 97.8% (95%CI = 96.3-98.8), positive predictive value was 91% (95%CI = 85.3-95), and negative predictive value was 42.6% (95%CI = 40-45.2). Raw seafood consumption in unvaccinated subjects was associated with the anti-HAV IgG positivity (OR = 2.1; 95%CI = 1.7-2.7; z = 7.4; P < 0.0001).The vaccination program seems to have changed the virus circulation pattern, with a higher seronegativity rate among subjects not included in the vaccination strategy: 67% of susceptible subjects were among younger people aged 27 to 35 years. Immunization program implementation with a catch-up strategy may be needed to avoid a possible increase of hepatitis A incidence and outbreaks in Apulia. Epidemiological surveillance should be continued and vaccination should actively be offer for free to all the cases contacts, in order to prevent new outbreaks onset.