Hepatitis B imposes a major public health problem with an increased risk of occupational exposure among unvaccinated health care workers. This study was conducted to determine the Hepatitis B vaccination status, along with the knowledge, attitude, and practice regarding Hepatitis B, among preclinical medical students of a medical college in Nepal. This descriptive study was conducted among preclinical students of a medical college in Kathmandu, Nepal from 6th July to 14th July 2020. The whole sampling technique was used. Data were collected using a pretested, self-administered questionnaire which was emailed to individuals and analyzed with the statistical package for social sciences version-22. A total of 181 students participated in the study out of 198, giving a response rate of 91.4%. Among the study participants, only 67 (37%) were fully vaccinated against Hepatitis B while 71 (39.2%) were never vaccinated. For the majority (74.6%) of the non-vaccinated participants, the main reason for not getting vaccination was a lack of vaccination programs. Half the study participants (n = 92, 50.8%) had good knowledge, attitude and practice regarding hepatitis B. The median knowledge, attitude and practice scores towards Hepatitis B were 61.00 (57.00-66.00), 20(18.00-21.00) and 21(19.00-23.00) respectively. The majority of preclinical medical students were not fully vaccinated against Hepatitis B and only half of them had acceptable knowledge, attitude and practice towards Hepatitis B, which makes them vulnerable to the infection. This might represent the situation of not only Nepal, but also all South Asian countries, and creates concern about whether students take the vaccination programs seriously. Since unavailability of vaccination program is the main cause of non-vaccination, we strongly recommend the provision of the Hepatitis B vaccination program to the preclinical medical students.