Adverse childhood experiences (ACEs) of a woman can lead to dysregulated hypothalamus-pituitary-adrenal (HPA) axis during pregnancy, which can in turn adversely affect her offspring HPA axis function. Choline and docosahexaenoic acid (DHA) are dietary factors with the potential to favorably modify the stress response system. The current study aimed to investigate whether maternal choline intake and DHA status moderate the effects of maternal ACEs exposure on maternal and infant HPA axes function. Participants were a sub-sample of the prospective longitudinal Alberta Pregnancy Outcomes and Nutrition (APrON) study consisting of 340 mothers and 238 infants. We collected data on maternal ACEs, maternal choline intake (24-hour dietary recall) and serum phospholipid DHA concentrations (at each trimester). Women self-collected saliva samples on two consecutive days (at waking, +30 min, 1100 h, and 2100 h) in each trimester to calculate the cortisol awakening response (CAR) and total daytime cortisol. Infants’ salivary cortisol was measured before and after (20, and 40 min) exposure to a blood draw stressor 3 months postpartum. During pregnancy, choline intake moderated (reduced) the association between maternal ACEs and CAR (β = −0.003; 95% CI −0.006, −0.003), but not total daytime cortisol. DHA status did not moderate the association between ACEs and CAR or total daytime cortisol. Choline intake also moderated (reduced) the association between maternal CAR and infant cortisol during a stress task (β = −0.0001; 95% CI −0.0002, −0.00003). Maternal DHA status revealed no modifying effects on these associations. Our findings suggest that maternal choline intake, but not DHA status, can buffer the associations between ACEs and maternal HPA axis, as well as maternal and infant HPA axes function.