Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease and is characterized by the progressive loss of upper and lower motor neurons, leading to paralysis and death. Accumulation of cholesterol in the central nervous system (CNS) has been reported to actively contribute to the disease progression in Alzheimer’s disease, Huntington’s disease, Spinocerebellar ataxia and more recently ALS. Cholesterol is essential for myelin compartment, but also for its functional and structural role in plasmatic membrane. However, in the CNS, cholesterol is synthetized in situ and is not able to freely cross the blood brain barrier (BBB). Cholesterol-24-hydroxylase (CYP46A1) allows the conversion of cholesterol to 24-hydroxycholesterol, able to cross the BBB, thus regulating cholesterol homeostasis. Furthermore, this enzyme is a key neuronal stress response such as oxidative stress or protein aggregation. Therefore, we hypothesized that CYP46A1 could be relevant for a therapy in ALS to target both familial and sporadic forms of ALS independently from their genetic origin. In the severe SOD1ᴳ⁹³ᴬ model, we overexpressed CYP46A1 using an AAVPHP.eB able to cross the BBB after intravenous injection. As a first step, we confirmed that the AAVPHP.eB viral vector has a specific tropism for the CNS and especially motoneurons. Secondary, we demonstrated a significant and prolonged motor rescue of animals treated pre or post-symptomatically, but also a preventive effect on myelin loss, compared to untreated animals. Evaluation of this therapeutic strategy is ongoing in another model of ALS.