Objectives: Since 1991 hepatitis B vaccination has been mandatory for all newborns in Italy. The aim of the study was to verify the long-term seroprevalence and the efficacy of hepatitis B vaccination in medical students of the University of Siena. Material and Methods: A cross-sectional observational study was conducted on a population of 850 medical students of the University of Siena (322 males and 528 females, mean age: 23 years) by obtaining from the medical reports the serological analysis data for the total anti-hepatitis B antibodies (HBsAb) and information on hepatitis B vaccination (number of vaccine doses, age at the first vaccination, time since the final vaccination dose, country of origin). Raw odds ratios (ORs) and 95% confidence intervals (CIs) were initially calculated to evaluate the association between 2 variables. The adjusted ORs were then calculated using a multivariate logistic regression model to study the association between the variables and the possible confounding factors. Results: Overall, 593 students (69.76%) were immunized against hepatitis B, while 257 (30.24%) had HBsAb antibody titer <10 mIU/ml. From the OR calculation, an inverse correlation emerged between seropositivity to hepatitis B and age, and between seropositivity to hepatitis B and the age at the first vaccination dose. There was also a correlation between seropositivity and the number of vaccination doses received. By performing the multivariate logistic analysis, correlations with these variables were confirmed. Conclusions: A significant part of the studied population was not immunized against hepatitis B virus, despite the fact that vaccination had been carried out as prescribed by law. The results of the study reaffirm the importance of health surveillance in subjects at biological risk such as medical students. Int J Occup Med Environ Health. 2022;35(1):75–80