Objective Prolonged maternal separation (PMS) in the first two weeks of life has been associated with poor growth with lasting effects in the brain structure and function. This study aimed to investigate whether PMS-induced undernutrition could cause systemic inflammation and changes in nutrition-related hormonal levels, affecting hippocampal structure and neurotransmission in C57BL/6J suckling mice. Methods This study assessed mouse growth parameters coupled with IGF-1 serum levels. In addition, leptin, adiponectin and corticosterone serum levels were measured following PMS. Hippocampal stereology and the amino acid levels were also assessed. Furthermore, we measured myelin basic protein and synapthophysin (SYN) expression in the overall brain tissue and hippocampal SYN immunolabeling. For behavioral tests, we analyzed the ontogeny of selected neonatal reflexes. PMS was induced by separating half the pups in each litter from their lactating dams for defined periods each day (4h on day 1, 8h on day 2, and 12h thereafter). A total of 67 suckling pups were used in this study. Results PMS induced significant slowdown in weight gain and growth impairment. Significant reductions in serum leptin and IGF-1 levels were found following PMS. Total CA3 area and volume were reduced, specifically affecting the pyramidal layer in PMS mice. CA1 pyramidal layer area was also reduced. Overall hippocampal SYN immunolabeling was lower, especially in CA3 field and dentate gyrus. Furthermore, PMS reduces hippocampal aspartate, glutamate, and GABA levels, as compared with unseparated controls. Conclusion Altogether, these findings suggest that PMS causes significant growth deficits and alterations in hippocampal morphology and neurotransmission.