Maternal plasma phospholipid polyunsaturated fatty acids (PUFAs) play critical roles in maternal health and fetal development. Beyond dietary factors, maternal moderate-to-vigorous physical activity (MVPA) has been linked to multiple health benefits for both the mother and offspring, but studies investigating the influence of maternal MVPA on maternal PUFA profile are scarce. The objective of present study was to examine the time-specific and prospective associations of MVPA with plasma PUFA profile among pregnant women. This study included 321 participants from the National Institute of Child Health and Human Development (NICHD) Fetal Growth Studies–Singletons cohort. Maternal plasma phospholipid PUFAs and MPVA were measured at four visits during pregnancy (10–14, 15–26, 23–31, and 33–39 gestational weeks (GW)). Associations of maternal MVPA with individual plasma PUFAs and desaturase activity were examined using generalized linear models. Maternal MVPA was associated inversely with plasma phospholipid linoleic acid, gamma-linolenic acid, and Δ6-desaturase in late pregnancy (23–31 or 33–39 GW), independent of maternal age, race, education, parity, pre-pregnancy body mass index, and dietary factors. Findings from this longitudinal study indicate that maternal habitual MVPA may play a role on PUFAs metabolism, particular by alerting plasma n-6 subclass and desaturase activity in late pregnancy. These associations are novel and merit confirmation in future studies.