Despite increasing interest in the health effects of polyunsaturated FAs (PUFAs), their roles in fetal and neonatal growth remain understudied. Within the NICHD Fetal Growth Studies-Singleton Cohort, we prospectively investigated the associations of individual and subclasses of plasma phospholipid PUFAs at gestational weeks (GW) 10-14, 15-26, 23-31, and 33-39 with neonatal anthropometric measures as surrogates for fetal growth among 107 women with gestational diabetes mellitus (GDM) and 214 non-GDM controls. Multivariable weighted linear regression models estimated the associations between plasma phospholipid PUFAs and neonatal anthropometric measures. Adjusted beta coefficients for phospholipid docosahexaenoic acid (DHA) per standard deviation (SD) increase at GW 23-31 in association with birthweight z-score, neonatal length, and neonatal fat mass were 0.25 (95% CI: 0.08-0.41), 0.57 (0.11-1.03) cm, and 54.99 (23.57-86.42) g, respectively; all false discovery rates (FDRs) < 0.05. Estimated Δ5-desaturase activity per SD increase at GW 33-39 but not at other time points was positively associated with birthweight z-score: 0.29 (95% CI: 0.08-0.33); neonatal length: 0.61 (0.29-0.94) cm; and neonatal fat mass: 32.59 (8.21-56.96) g; all FDRs < 0.05. Longitudinal analysis showed consistent results. Our findings suggest that mid-to-late pregnancy presented as critical windows for primarily diet-derived DHA and Δ5-desaturase activity in relation to neonatal anthropometric measures.