Background This study aimed to investigate the factors affecting ambivalent feelings among women who gave birth after having received negative results on non-invasive prenatal genetic testing (NIPT). Methods A questionnaire was sent to women who received a negative NIPT result, and a contents analysis was conducted for those 1562 women who responded to the open-ended question. The contents of these qualitative data were analyzed using the N-Vivo software package. Results Environmental factors, genetic counseling-related factors, and increased anticipatory anxiety affected the feeling of ambivalence among pregnant women. Furthermore, pregnant women desired more information regarding the detailed prognosis for individuals with Down syndrome and living with them and/or abortion, assuming the possibility that they were positive. Conclusions Three major interrelated factors affected the feeling of ambivalence in women. Highlighting and discussing such factors during the genetic counseling may resolve some of these ambivalences, thereby enhancing the quality of decisions made by pregnant women.