Background: N-3 polyunsaturated fatty acids (n-3 PUFA) may have multiple beneficial effects on the outcome of pregnancy, maternal health and child development. The present study introduced the protocol of a birth cohort study to examine the beneficial effects of n-3 PUFA status in pregnant Japanese women as an adjunct study of the Japan Environment and Children’s Study (JECS). Methods: The JECS participants in the coastal areas of Miyagi Prefecture were further invited to participate in this adjunct study, and 1,878 pregnant women were enrolled prior to delivery. Their n-3 PUFA status was evaluated with fatty acid profiles in erythrocytes of maternal blood collected from 1,623 mothers at 24–30 weeks of gestation and cord blood from 1,505 deliveries. Results: The baseline results, including comprehensive data on the fatty acid status and determinants affecting the PUFA status, were analyzed. In stepwise multivariate analyses, the cord blood docosahexaenoic acid (DHA) level was found to be significantly influenced by the DHA level in maternal blood, the child’s sex, and the gestational period. The maternal DHA level was influenced by fish intake, maternal age, and the prepregnancy body mass index. While cord blood eicosapentaenoic acid (EPA) was influenced by maternal EPA, fish intake, and season at birth, additional factors such as maternal education, household income, and smoking habits affected the maternal EPA content. Conclusion: Further studies are warranted to clarify the nutritional impacts of n-3 PUFA in pregnant Japanese women of the cohort study.