Seroepidemiological studies on pertussis, diphtheria and tetanus were performed on 1,540 nursing students enrolled in S-university between 1994 and 2011. Antibody titers against pertussis toxin (PT) and filamentous hemagglutinin (FHA), diphtheria antitoxin titer, and tetanus antitoxin titer were measured using sera taken during enrollment. The antibody-seropositive rates and geometric mean titers (GMTs) were calculated by according to birth year (1975-1993). The pertussis anti-PT and anti-FHA antibody-seropositive rates (seropositive levels were both defined as ≥10 EU/mL) were 12%-53% and 47%-84%, respectively. The anti-PT antibody-seropositive rate was lower than the anti-FHA rate. The anti-PT antibody GMTs were 2.0-11 EU/mL, whereas the anti-FHA antibody GMTs were 10 EU/mL or more (8.8-31 EU/mL) in almost every group. The diphtheria antitoxin-seropositive rate (≥0.1 IU/mL) was 49%-79%, and the GMT was nearly 0.1 IU/mL. The tetanus antitoxin-seropositive rate (≥0.01 IU/mL) was 91%-100%, and the GMT was 0.3 IU/mL or more for all the groups. While the nursing students' diphtheria and tetanus antitoxin levels were sufficient to prevent both diseases, the anti-PT antibody-seropositive rate and the GMT were both lower than the level required to prevent pertussis, suggesting that many of the nursing students were pertussis-susceptible. These findings suggest a need for pertussis vaccination in young adults or early adolescents.