We evaluated the seroprevalence of vaccine-preventable infectious diseases among Japanese healthcare students to create immunization guidelines. Between 2007 and 2012, a total of 1746 Japanese medical, nursing, and paramedical students were serologically screened for measles, mumps, rubella, varicella, and hepatitis B virus (HBV) antibodies at the time of admission. In 2007, the seroprevalence of measles and mumps was 52.7% and 65.6%, respectively. The seroprevalence of measles dramatically increased to 96.6% in 2009 and was then sustained at >90%. The seroprevalence of mumps gradually increased to >80.0% between 2010 and 2012. The seroprevalence of rubella remained at >90% except in 2008 (85.6%), and the seroprevalence of varicella was sustained at >92% throughout 2007-2012. The seroprevalence of HBV antibody remained at <7% during 2007-2012. Although the seroprevalence of vaccine-preventable infectious diseases among Japanese healthcare students increased during the 2007-2012 study period, a substantial number of students were susceptible to vaccine-preventable infectious diseases. Therefore, we propose targeted immunization of Japanese healthcare students using serological screening prior to clinical training.