Activating transcription factor 3 (ATF3), a member of the ATF/cAMP response element binding (CREB) family, is upregulated by various intracellular and extracellular signals such as injury and signals related to cell proliferation. ATF3 also belongs to the regeneration-associated genes (RAG) group of transcription factors. RAG and ATF/CREB transcription factors that play an important role in embryonic neuronal development and PNS regeneration may also be involved in postnatal neuronal differentiation and development, as well as in the regeneration of the injured CNS. Here we investigated the effect of ATF3 in differentiation, neural outgrowth, network formation, and regeneration after injury using postnatal dissociated cortical neurons derived from neonatal opossums (Monodelphis domestica). Our results show that RAG and ATF genes are differentially expressed in early differentiated neurons versus undifferentiated neurospheres and that many members of those families, ATF3 in particular, are upregulated in cortical cultures obtained from younger animals that have the ability to fully functionally regenerate spinal cord after injury. In addition, we observed different intracellular localization of ATF3 that shifts from nuclear (in neuronal progenitors) to cytoplasmic (in more mature neurons) during neuronal differentiation. The ATF3 inhibition, pharmacological or by specific antibody, reduced the neurite outgrowth and differentiation and caused increased cell death in early differentiating cortical neuronal cultures, suggesting the importance of ATF3 in the CNS development of neonatal opossums. Finally, we investigated the regeneration capacity of primary cortical cultures after mechanical injury using the scratch assay. Remarkably, neonatal opossum-derived cultures retain their capacity to regenerate for up to 1 month in vitro. Inhibition of ATF3 correlates with reduced neurite outgrowth and regeneration after injury. These results indicate that ATF3, and possibly other members of RAG and ATF/CREB family of transcription factors, have an important role both during cortical postnatal development and in response after injury.