BACKGROUND AND PURPOSE Human dihydroorotate dehydrogenase (DHODH) represents a promising therapeutic target for chronic inflammatory and autoimmune diseases. The aim of this study was to discover novel DHODH inhibitor and evaluate the potential of DHODH inhibition in treating multiple sclerosis (MS), a popular chronic inflammatory disease of the central nervous system. EXPERIMENTAL APPROACH Biochemical and Biophysical methods, including enzymatic kinetic analysis, thermofluor assay, isothermal titration calorimetry and X-ray crystallography were used to assess DHODH inhibition. The immunomodulatory activity was assessed by using concanavalin a-triggered T-cell assay and mixed lymphocyte reaction assay. MOG-induced experimental allergic encephalomyelitis (EAE) was used to assess the in vivo therapeutic effects. Myelin destruction and blood–brain barrier (BBB) was evaluated via in vivo imaging KEY RESULTS Piperine was identified as a natural inhibitor of human DHODH with an IC50 value of 0.88 ± 0.04 μM. In addition, we resolved the co-complex crystal structure of DHODH and piperine at 1.98 Å resolution and found that Tyr356 residue of DHODH is critical for piperine binding. Moreover, piperine can markedly suppress T cell overactivation via a DHODH dependent-manner. Finally, we found that piperine exhibits strong preventive and therapeutic effect in the MOG-induced EAE by restricting inflammatory cells infiltration into the CNS and by preventing myelin destruction and blood–brain barrier (BBB) disruption. CONCLUSION AND IMPLICATIONS Taken together, these findings highlight DHODH as a therapeutic target for autoimmune disease of the nervous system, and demonstrate a novel pharmacological role for piperine in the treatment of MS.