Objectives: While women are expected to support Japan’s labor force, they often quit their jobs because of pregnancy and childbirth. Occupational stress affects reproductive life events unique to women, including pregnancy intention and premature birth. This study aimed to examine occupational stress and its related factors among working women of reproductive age, especially from the perspective of pregnancy. Methods: This cross-sectional observational study involved a web survey of childless female workers aged 20–44 years, conducted in April–May 2019 in Japan. The survey items included the Brief Job Stress Questionnaire, demographic characteristics, and work-related variables. Logistic regression analysis was performed with physical and psychological stress reactions as dependent variables and demographic characteristics and work situation as control variables. Results: Of the 904 employees, 450 were pregnant. Physical stress reactions indicated that being pregnant and stress risk related to job control, interpersonal conflict, suitable jobs were significantly associated. Further, psychological stress reactions including stress risk related to job overload, job control, interpersonal conflict, and suitable jobs were significantly associated, but being pregnant was not. Among pregnant women, being in the first trimester and maternity harassment were significantly associated with physical and psychological stress reactions. Conclusions: To ensure that women in their prime continue working through pregnancy and after childbirth, gender-friendly social systems and workplaces free of harassment are required, as well as workplace support systems appropriate for each stage of pregnancy.