AbstractPrenatal exposure to maternal depression and serotonin reuptake inhibitor (SRI) antidepressants both affect the development of the hypothalamic–pituitary–adrenal (HPA) system, possibly via the neurotransmitter serotonin (5HT). In a community cohort, we investigated the impact of two factors that shape prenatal 5HT signaling (prenatal SRI [pSRI] exposure and child SLC6A4 genotype) on HPA activity at age 6 years. Generalized estimating equation (GEE) models were used to study associations between cortisol reactivity, pSRI exposure, and child SLC6A4 genotype, controlling for maternal depression, child age, and sex (48 pSRI exposed, 74 nonexposed). Salivary cortisol levels were obtained at five time points during a laboratory stress challenge: arrival at the laboratory, following two sequential developmental assessments, and then 20 and 40 min following the onset of a stress‐inducing cognitive/social task. Cortisol decreased from arrival across both developmental assessments, and then increased across both time points following the stress challenge in both groups. pSRI‐exposed children had lower cortisol levels across all time points. In a separate GEE model, we observed a lower cortisol stress response among children with L_G/S alleles compared with children with La/La alleles, and this was particularly evident among children of mothers reporting greater third trimester depressed mood. Our findings suggest that pSRI exposure and a genetic factor associated with modulating 5HT signaling shaped HPA reactivity to a laboratory stress challenge at school age.