Fluctuations in ovarian steroids across the estrous and menstrual cycle in female rats and women, respectively, are associated with changes in anxiety. Pregnancy causes long-term changes to ovarian hormone release, yet research on estrous- and menstrual-related changes in anxiety has focused on reproductively inexperienced females. Therefore, this study assessed whether the impact of estrous and menstrual cycles on anxiety differs pre- versus post-motherhood in female rats (n= 32) and a community sample of women (n= 63). Estrous cycle phase altered anxiety-like behavior in virgin rats, but had no effect in age-matched mother rats tested 1-month post-weaning. In humans, menstrual cycle phase was associated with ecological momentary assessed anxiety and mood in non-mothers, but not mothers; although, the menstrual cycle × reproductive status interaction for anxiety, but not mood, was rendered non-significant with age and cycle length as covariates. These findings suggest that changes in anxiety coincident with cycling hormones is an evolutionarily conserved feature of the estrous and menstrual cycle in rats and women, which is mitigated following motherhood in both species. We identified several potential mechanisms for the observed dissociation in estrous cycle effects on anxiety. Compared to virgin rats, mother rats had a lower peak and blunted decline in circulating allopregnanolone during proestrus, upregulated GABAAreceptor subunit (α1, α2, α5, α4, ß2) mRNA in the ventral hippocampus, and altered corticotropin-releasing hormone mRNA across the estrous cycle in the basolateral amygdala. Together, these findings suggest that the mechanisms underlying anxiety regulation undergo fundamental transformation following pregnancy in female rats and humans.