Circulating individual SFAs in pregnant females are critical for maternal and fetal health. However, research on identifying their modifiable factors is limited. We aimed to examine the associations of total physical activity (PA) and types of PA with circulating individual SFAs during pregnancy in a multiracial/multiethnic cohort of pregnant females in the United States. The study included participants in a nested case-control study (n = 321) from the Eunice Kennedy Shriver NICHD Fetal Growth Studies-Singleton Cohort. Sampling weights were applied, so the results represented the entire Fetal Growth Cohort. Plasma phospholipid SFAs were measured at 4 visits [10-14 (visit 1), 15-26 (visit 2), 23-31 (visit 3), and 33-39 (visit 4) weeks of gestation] throughout pregnancy. PA of the previous year at visit 1 and since the previous visit at the subsequent visits was assessed using the validated Pregnancy PA Questionnaire. Time-specific and longitudinal associations were examined using multivariable linear and generalized estimating equation models. Total PA (metabolic equivalent of task-h/wk) was positively associated with circulating heptadecanoic acid (17:0) at visit 1 (β × 103: 0.07; 95% CI: 0.02, 0.11) and pentadecanoic acid (15:0) at visit 3 (β × 103: 0.09; 95% CI: 0.03, 0.14) independent of sociodemographic, reproductive, pregnancy, and dietary factors. Across the 4 visits, the positive associations with total PA were consistent for pentadecanoic acid (β × 103: 0.06; 95% CI: 0.02, 0.10) and heptadecanoic acid (β × 103: 0.10; 95% CI: 0.06, 0.14). Out of the 4 PA types (i.e., sports/exercise, household/caregiving, transportation, and occupational PA) considered, the magnitude of positive associations was the largest for sports/exercise PA. Our findings suggest that maternal PA is positively associated with circulating pentadecanoic and heptadecanoic acids. The findings warrant confirmation by future studies.This trial was registered at clinicaltrials.gov as NCT00912132.