<p>Demyelination is a common feature of numerous neurological disorders including multiple sclerosis and leukodystrophies. Although myelin can be regenerated spontaneously following injury, this process is often inadequate, potentially resulting in neurodegeneration and exacerbating neurological dysfunction. Several drugs aimed at promoting the differentiation of oligodendrocyte precursor cells (OPCs) have yielded unsatisfactory clinical effects. A recent study has shifted the strategy of pro-OPC differentiation towards enhancing myelinogenesis. In this study we identified the pro-myelinating drug using a zebrafish model. Five traditional Chinese medicine monomers including gastrodin, paeoniflorin, puerarin, salidroside and scutellarin were assessed by bath-application in Tg&#xa0;(MBP:eGFP-CAAX) transgenic line at 1–5 dpf. Among the 5 monomers, only gastrodin exhibited significant pro-myelination activity. We showed that gastrodin (10 µM) enhanced myelin sheath formation and oligodendrocyte (OL) maturation without affecting the number of OLs. Gastrodin markedly increased the phosphorylation levels of PI3K, AKT, and mTOR in primary cultured OLs via direct interaction with PI3K. Co-treatment with the PI3K inhibitor LY294002 (5 µM) mitigated gastrodin-induced OL maturation. Furthermore, injection of gastrodin (100 mg·kg⁻¹·d⁻¹, i.p.) effectively facilitated remyelination in a lysophosphatidylcholine-induced demyelinating mouse model and alleviated demyelination in the experimental autoimmune encephalomyelitis mice. These results identify gastrodin as a promising therapeutic agent for demyelinating diseases and highlight the potential of the zebrafish model for screening pro-myelinogenic pharmacotherapy.</p>