Maternal perinatal stress (MPS) is increasingly recognized as a determinant of early socioemotional development. This study examined whether MPS predicts infant temperament at three months and explored the mediating role of maternal NR3C1 gene expression. A cohort of 198 mother–infant dyads was assessed through validated psychological questionnaires, molecular analyses, and caregiver-reported temperament measures across pregnancy and early postpartum. Results showed that elevated MPS, particularly during the second trimester, predicted lower infant surgency, higher negative affectivity, and reduced effortful control. Regression models revealed that second-trimester stress was the strongest predictor of infant temperament dimensions, explaining up to 47% of variance. MPS was also associated with reduced maternal NR3C1 expression, which in turn predicted lower infant surgency. Mediation analysis confirmed that maternal NR3C1 expression significantly mediated the link between cumulative perinatal stress and infant surgency. These findings provide novel evidence on molecular pathways underlying the developmental origins of health and disease (DOHaD), suggesting that maternal stress during pregnancy may become biologically embedded and shape infant behavioral phenotypes. Implications for early prevention strategies and perinatal mental health policies are highlighted.