Despite the recent increase in infertility and perinatal complications, preconception care is not commonly available in Japan. Working women are considered to have the greatest need for preconception care, as increasingly they marry and have children later in life. This study aimed to assess the feasibility and effectiveness of preconception check-ups in the workplace. We provided 51 female employees aged 18-39years with free preconception check-ups, including additional blood tests and an online medical questionnaire, during mandatory health check-ups at their workplace. A doctor provided online counseling based on the check-up results. We assessed fertility knowledge using the Cardiff Fertility Knowledge Scale (CFKS-J) and childbearing desire pre- and postintervention. Preconception check-ups revealed various potential risk factors for future pregnancies, including underweight (12%), obesity (20%), Chlamydia trachomatis IgG antibody positivity (22%), low rubella IgG antibody levels (47%), iron deficiency (12%), and 25-hydroxyvitamin D levels <30ng/mL (98%). Postintervention, the participants reported high satisfaction with the check-ups and significantly advanced their reproductive plans (P = .008). Furthermore, 95% of the participants indicated an intention to seek medical attention or make lifestyle changes. The postintervention CFKS-J score (mean [SD]) was higher than the preintervention score (71.7 [19.3] vs 63.0 [22.0]; P = .006). We developed a preconception check-up package that can be integrated into workplace health examinations, complemented by tailored counseling. This novel check-up package is a feasible and effective approach for improving preconception health and fertility awareness.