Multiple sclerosis (MS) is a complicated inflammatory neurodegenerative disease of the central nervous system. Previous studies have demonstrated mitochondrial dysfunction in MS cortical pathology. In the present study, we showed that neuronal mitochondrial metabolite NAA, an indication of neuronal mitochondrial dysfunction, was decreased in MS gray matter. Decreased NAA was correlated with decreased acetate in adjacent white matter for the synthesis of myelin lipids. This suggested that one potential mechanism to reduce demyelination in MS was to restore neuronal mitochondrial function. In neuronal cell cultures, we observed that unexpected expression of neuronal hemoglobin increased cellular glycolysis rates, mitochondrial respiration rates and complex V levels. Hemoglobin alleviated respiration inhibition by excess NO. Neuronal hemoglobin interacted with nNOS in different brain regions responsible for learning and memory. Confocal microscopy specified the interaction between hemoglobin and nNOS to neuronal cell body, neurites, and synapses, which indicated that hemoglobin might regulate synaptic transmission by NO-mediated pathway. Additionally, hemoglobin levels were affected by conditions mimicking MS gray matter pathology in primary neurons. These findings showed implication of developing neuroprotective therapies for MS by increasing neuronal hemoglobin levels. The regulation between mitochondrial dysfunction and demyelination is reciprocal. Demyelination results in increased levels of free MBP in the lesion areas and the CSF of MS patients. In our SH-SY5Y neuroblastoma cell cultures, free MBP led to PS translocation to outer leaflet of plasma membrane, which might be a signal for microglial activation. Free MBP also decreased mitochondrial membrane potential in neuroblastoma cells, which might accumulate mitochondrial stress for mitochondrial dysfunction.