Jingzhaotoxin-XII (JZTX-XII), a 29-residue polypeptide, was purified from the venom of the Chinese tarantula Chilobrachys jingzhao. Electrophysiological recordings carried out in Xenopus laevis oocytes showed that JZTX-XII is specific for Kv4.1 channels, with the IC50 value of 0.363 microM. It interacts with the channels by modifying the gating behavior. JZTX-XII shares 80% sequence identity with phrixotoxin1, a potent inhibitor for Kv4.2 and Kv4.3 channels. Structure analysis indicates that the difference of the charge distribution in the interactive surface perhaps influences the specific pharmacology of the toxins. JZTX-XII should be a valuable tool for the investigation of the Kv4.1 channels.