Some studies demonstrate the beneficial role of high doses of vitamin B12 (VitB12) supplementation for the recovery from specific neurological impairments; however, the detailed mechanisms are not clearly elucidated. With this in mind, we employed an in vitro cell model made of hydrogen peroxide-insulted differentiated-SH-SY5Y (exhibiting a neuron-like phenotype) to study the effect of both a high and the physiological dose of VitB12 during recovery. The physiological dose of VitB12 showed an improvement in the cell count (although cell viability, evaluated by MTT, was comparably decreased in all conditions), as well as neurite elongation and maturation, as demonstrated by the evaluation of Growth Associated Protein-43 expression. Furthermore, mitochondrial morphology analysis revealed an increase in mitochondrial fusion only when the recovery was performed with the physiological concentration of VitB12. The changes in both mitochondrial morphology and cell viability induced by the physiological dose of VitB12 suggest a modification in the metabolic activity. To further investigate this, we performed NMR metabolomics. Additionally, due to the direct link between VitB12, metabolism and aberrant lipid incorporation in membranes, we also characterized chemical alterations at the cell surface using Raman analysis. Overall, our in vitro studies highlight a greater beneficial effect of the physiological dose of VitB12 for neuronal recovery compared to a high dose, thereby laying the foundation for future studies and medical interventions with the supplementation of the optimum dose of VitB12.