AbstractClC-2 transports chloride ions across plasma membranes and plays critical roles in cellular homeostasis. Its dysfunction is involved in diseases including leukodystrophy and primary aldosteronism. AK-42 was recently reported as a specific inhibitor of ClC-2. However, experimental structures are still missing to decipher its inhibition mechanism. Here, we present cryo-EM structures of apo ClC-2 and its complex with AK-42, both at 3.5 Å resolution. Residues S162, E205 and Y553 are involved in chloride binding and contribute to the ion selectivity. The side-chain of the gating glutamate E205 occupies the putative central chloride-binding site, indicating that our structure represents a closed state. Structural analysis, molecular dynamics and electrophysiological recordings identify key residues to interact with AK-42. Several AK-42 interacting residues are present in ClC-2 but not in other ClCs, providing a possible explanation for AK-42 specificity. Taken together, our results experimentally reveal the potential inhibition mechanism of ClC-2 inhibitor AK-42.