Docosahexaenoic acid (DHA), an essential n-3 long-chain polyunsaturated fatty acid (LCPUFA) abundant in fish, is crucial for infant brain development. We investigated the associations between maternal dietary habits, infant feeding patterns, and serum levels of DHA and other LCPUFAs in infants aged 5–6 months in Japan, where fish consumption is high. This cross-sectional study used serum samples from 268 infants enrolled in a randomized clinical trial. The frequency of mothers’ consumption of 38 food items and infant feeding patterns were prospectively surveyed. Cow’s milk formula (CMF) supplemented with 15.9% linolenic acid, 1.6% α-linolenic acid, 0.40% DHA, and 0.27% arachidonic acid was used. Significant positive associations with infants’ serum DHA levels were found for “Blue-back fish” (rho = 0.24; p = 0.0001) and “White fish” (rho = 0.25, p = 0.0001). The combined variable “Blue-White fish” was found to be significantly associated with higher serum DHA levels in infants (rho = 0.29, p < 0.0001). Predominantly breastfed infants had significantly higher serum DHA levels than those fed more CMF (rho = 0.32, p < 0.0001). After multivariate analysis, “Blue-White fish” and “Feeding patterns” remained significantly and independently associated with serum DHA levels. These findings suggest that frequent consumption of “Blue-back fish” and/or “White fish” by lactating mothers, along with prioritizing breastfeeding over DHA-supplemented CMF, might effectively increase infants’ serum DHA levels.