One of the major challenges of modern neurobiology concerns the inability of the adult mammalian central nervous system (CNS) to regenerate and repair itself after injury. It is poorly understood why regenerative potential is lost with evolution and development and why it becomes very limited in adult mammals. Even though our understanding of molecular and cellular mechanisms that promote or inhibit neuronal regeneration is expanding, it is still unclear what are the key differences between the neuronal systems that can and cannot regenerate, and how they can be manipulated to change the outcome of the injury. Our incomplete understanding of molecular events underlying neuronal development and regeneration is the main reason why neurodegenerative diseases and brain and spinal cord injuries are still mostly incurable today. A preferred model to study and reveal the cellular and molecular basis of regeneration is the neonatal opossum (Monodelphis domestica). Opossums are marsupials that are born very immature with the unique possibility to successfully regenerate spinal cord after injury in the first two weeks of their life. After that, the regenerative capacity is abruptly lost: at 14 days in cervical spinal segments and at 17 days in less mature lumbar spinal segments. In this thesis, we used neonatal opossums to study molecular and cellular properties of spinal tissue that has and does not have the capacity to regenerate after injury, to pinpoint the key differences. Using a comparative proteomic approach, for the first time, we identified the proteins unique and differentially distributed in the intact opossum spinal tissue with different regenerative capacities. We have identified a total number of 4735 proteins involved in various cellular processes such as cell growth, proliferation, differentiation, transcription, cell signaling, cytoskeleton and extracellular matrix organization, axon guidance molecules, neurotrophic factors and entire intracellular molecular pathways like mTOR and MAPK signaling pathway. We have also identified many myelin associated proteins, known to act as inhibitors of CNS axon regeneration, which was the positive control for our overall experimental procedure. Most interestingly, we have identified a number of proteins related to different neurodegenerative diseases that change in the opossum spinal cord during the period critical for neuroregeneration. The different distribution of the selected proteins detected by comparative proteomics was further confirmed by Western blot, and the changes in the expression of related genes were analyzed by quantitative reverse transcription PCR. Furthermore, we explored the cellular localization of the selected proteins using immunofluorescent microscopy. We showed that during the period of development when viii regeneration stops being possible, the substantial number of proteins known to be involved in regeneration and development change their level in the opossum spinal cord. These results upgraded the previous transcriptomic data about the genes differentially expressed in regenerating and non-regenerating opossum spinal cord tissue. Comparison of the data obtained by genomic and proteomic approaches allowed the identification of molecules of interest and narrowed the number of candidates for functional assays. For the first time, we successfully established primary neuronal spinal cell cultures from neonatal opossums of different ages and different regenerative capacities, which represent a novel mammalian in vitro platform, particularly useful to study CNS development and regeneration, and to test the functional role of the candidate molecules, together with the intact neonatal opossum spinal cord preparation. We have also developed the neuroregeneration scratch test to be performed on primary neuronal spinal cell cultures deriving from P5 opossums. Taken together, the results of this thesis contribute to a better understanding of neuronal regeneration in mammals and identify candidate molecules as future targets to promote neuroregeneration in mammalian CNS.