Early-life stress elicits anxiety and depressive-like behaviors both in humans and in rodents. At the same time, serotonin deficits have been linked to the origin of these mental disorders, and drugs enhancing brain serotonin levels are the first line treatment for such neuropsychiatric conditions. Here we sought to investigate the role of serotonin in a model of early-life stress in mice. C57BL/6 pups were subjected to the maternal separation protocol (3h/day) during a postnatal critical period (P2-P14) while receiving daily injections of the tryptophan hydroxylase inhibitor para-chlorophenylalanine (PCPA, 10mg/kg/day s.c.). We analyzed the synaptic innervation of the prefrontal cortex to the dorsal raphe nucleus (PFC-to-DRN) circuit by high-resolution microscopy (array tomography), and serotonin cell activation by c-fos immunohistochemistry. Lastly, emotional responses to stress were evaluated in a battery of behavioral paradigms at adulthood (from P80). Our study showed that male mice were more susceptible to stress, resulting in long-lasting anxiety and depressive-like behaviors. PFC-to-DRN circuit connectivity alterations by stress were prevented by depletion of serotonin, and DRN responses to an acute stress were altered in mice receiving PCPA. Our results emphasize the emotional alterations caused by early-life stress and the interaction with the serotonin transmission, in the search for understanding a main risk factor for the development of psychiatric disorders.