While chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K+ current IK1 and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac IK1. The voltage- and K+-dependence of chloroquine block implied that the binding site was located within the ion conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine binding site within the cytoplasmic pore domain, rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ion channel blockers, chloroquine does not bind within the transmembrane pore. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block IK1 even in the presence of high affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1 blocking properties.