Methotrexate (MTX) has been widely used for the treatment of many types of autoimmune diseases, such as rheumatoid arthritis, psoriasis and dermatomyositis. However, its pharmacological mechanism is still unclear completely. In this study, we found that MTX is a potent and selective inhibitor of the Kv1.3 channel, a class of potassium channels highly associated with autoimmune diseases. Electrophysiological experiments showed that MTX inhibited human Kv1.3 channel with an IC50 of 41.5 +/- 24.9 nM, and 1 mu M MTX inhibited 32.6 +/- 1.3% and 25.6 +/- 2.2% of human Kv1.1 and Kv1.2 channel currents, respectively. These data implied the unique selectivity of MTX towards the Kv1.3 channel. Excitingly, using channel activation and chimeric experiments, we found that MTX bound to the outer pore region of Kv1.3 channel. Mutagenesis experiments in the Kv.3 channel extracellular pore region further showed that the Dsp371, Thr373 and His399 residues of outer pore region of Kv1.3 channel played important roles in MTX inhibiting activities. In conclusion, MTX inhibited Kv1.3 channel by targeting extracellular pore region, which is different form all the report small molecules, such as PAP-1 and 4-AP, but similar with many natural animal toxin peptides, such as ChTX, ShK and BmKTX. To the best of our knowledge, MTX is the first small molecular scaffold targeting the Kv1.3 channel extracellular pore region, suggesting its potential applications for designing novel Kv1.3 lead drugs and treating Kv1.3 channel-associated autoimmune diseases. (C) 2020 Elsevier Inc. All rights reserved.