As potentially the most common cause of neurological disability in young adults, multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that results in chronic progressive disability for most patients. None of existing therapeutic agents could effectively inhibit the chronic progression, primarily due to their incapacity to stop or reverse remyelination failure in demyelinating lesions. Identifying compounds that promote remyelination represents a major challenge in the development of therapeutics for MS. Based on the drug-repurposing strategy and signature mapping approach, we employed the expanded Connectivity Map (CMap) and in cell western analysis to efficiently screen potential compounds. Ipratropium was ultimately selected and further validated for the efficacy in modulating OPCs differentiation in vitro and myelin regeneration in the demyelinating models. Collectively, our results provide a novel high-throughput screening strategy for potential regenerative therapeutics in MS.