Depression and anxiety are leading causes of disability worldwide, yet their developmental origins remain unclear. To explore early mechanisms of vulnerability to psychiatric disorders, we used a mouse model of adult emotional vulnerability induced by the early postnatal exposure to the antidepressant fluoxetine (FLX). C57BL/6 mice (both sexes) received FLX (10 mg/kg/day, p.o.) in 3% sucrose from postnatal day (P)2 to P14. At P15, we investigated the early impact on the prefrontal cortex-to-dorsal raphe nucleus (PFC-DRN) circuit, which is implicated in stress coping and mood regulation. Using the high-resolution microscopy technique, Array Tomography, we observed a selective ~40% increase in glutamatergic PFC inputs to DRN serotonin (5-HT) neurons. Ex-vivo patch-clamp recordings supported the presence of additional functional glutamatergic synapses. Following acute stress in the forced swim test (FST), c-fos immunohistochemistry and layer-specific markers revealed heightened activation of specific PFC projection-neurons and increased 5-HT1A receptor-mediated inhibition in the DRN. Behaviorally, FLX-exposed mice showed reduced immobility in the FST, an effect reversed by 5-HT1A receptor blockade using the selective antagonist WAY-100635. Altogether, these findings reveal that postnatal FLX induces structural and functional remodeling of the nascent PFC-DRN circuit, likely contributing to altered stress responses and emotional behavior later in life.