This study examined the association of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intake during the second trimester with low birth weight (LBW) in pregnant Japanese women and was conducted in conjunction with the Japan Pregnancy Eating and Activity Cohort (J-PEACH) study. The study included 504 pregnant women from four Japanese sites. During the second trimester (14–27 weeks), the participants filled out a self-administered questionnaire assessing the frequency of DHA and EPA supplement intake in the past month, as well as a brief-type self-administered diet history questionnaire (BDHQ). The analysis involved data from two time points: responses to the BDHQ and infant data at birth. In total, 471 and 33 participants were classified into the normal birth weight and LBW groups, respectively. The participants were divided into high-, medium-, and low-intake groups based on their total dietary and EPA and DHA supplementary intakes. The Cochran–Armitage trend test was used to analyze the data; the prevalence of LBW was higher in the low-intake group (p = 0.04). There was no significant sex-based trend (p = 0.27 and p = 0.35). In Japanese women, low dietary and supplementary EPA and DHA intake until the second trimester were risk factors for LBW.