The use of prenatal testing in Japan is expected to increase. However, there are ethical concerns regarding pregnancy termination upon the detection of fetal chromosomal abnormalities, such as Down syndrome. Furthermore, factors associated with decision-making following a positive result of Down syndrome after prenatal screening remain unclear. Therefore, we aimed to evaluate the association between decision-making among university students following a hypothetical positive result of Down syndrome in prenatal screening and their perception of their future career development defined as vocational identity. This cross-sectional study included 256 individuals (109 men, 143 women, and four who preferred not to answer). Self-anonymous semi-structured questionnaires were distributed to collect information regarding socio-demographic characteristics, vocational identity, and decision-making following a positive prenatal screening result of Down syndrome. Vocational identity was assessed using the Vocational Identity Measure. Women students were more likely to intend to continue the pregnancy (76.9%, p < 0.05); however, students without siblings (68.2%, p < 0.01) and men and women students with higher scores for vocational identities who were raised in an academically oriented family were more likely to intend to terminate the pregnancy (p < 0.05). Therefore, gender and vocational identity were associated with decision-making following a positive result of Down syndrome. Further qualitative and quantitative studies on factors associated with decision-making following a positive result of Down syndrome are necessary to eliminate the burden and social barrier, including gender division of labor and the effect of vocational career for people wishing to parent a child with Down syndrome.