Although preventable with vaccination, Hepatitis B virus (HBV) infection is a major health concern, with »400 million people at risk of developing the chronic form of the disease worldwide. The anti-HBV vaccine consists of a recombinant HBV surface antigen (HBsAg), which induces specific anti-HBs antibodies and confers 95% protection for >20 y. The aim of the present study was to analyze the response to HBV vaccination by measuring anti-HBs antibodies in serum samples from medical students of a public university in Puebla, Mexico. HBV infection markers HBsAg and anti-HBs, were also determined. A total of 201 students were included and vaccination coverage was found at 54%. Overall seropositivity for HBsAg, anti-HBc and anti-HBs determined by ELISA was 0.5%, 1.0% and 47%, respectively. Protective levels of anti-HBs >10 mIU/mL were found in 93.2% of subjects vaccinated with 2 or 3 doses and in 40% of those vaccinated with a single dose; while only 4.8% of unvaccinated subjects were anti-HBs positive. The response to the HBV vaccine was different in each participant, despite similar vaccination scheme. A history of blood transfusion/organ transplant or more than 2 sexual partners was significantly associated with anti-HBc positivity, OR D 399 (p D 0.010) and OR D 19.9 (p D 0.044), respectively. HBV immunization coverage was low in our sample compared with reports from countries with similar HBV prevalence, but anti-HBs in vaccinated individuals were in the expected range. It is important to promote HBV vaccination and awareness among medical students, due to their exposure risk.