Background: The acetylcholine activated inward rectifier potassium current (IKACh) is constitutively active in persistent atrial fibrillation (AF). We tested the hypothesis that blocking IKACh with the small molecule chloroquine terminates persistent AF.Methods: We used a sheep model of tachypacing induced persistent AF, molecular modeling, electrophysiology, and structural biology approaches.Results: The IC50 of IKACh block with chloroquine, measured by patch clamp, was 1 μM. In optical mapping of sheep hearts with persistent AF, 1 μM chloroquine restored sinus rhythm. Molecular modeling suggested that chloroquine blocked the passage of a hydrated potassium ion through the intracellular domain of Kir3.1 (a molecular correlate of IKACh) by interacting with residue F255, in close proximity to I228, Q227, and L299. H N HSQC of purified Kir3.1 intracellular domain confirmed the modeling results. F255, I228, Q227, and L299 underwent significant chemical shift perturbations upon drug binding. We then crystallized, and solved a 2.5Å X-ray structure of Kir3.1 with F255A mutation. Modeling of chloroquine binding to the mutant channel suggested that the drug’s binding to the pore becomes off-centered, reducing its ability to block a hydrated potassium ion. Patch clamp validated the structural and modeling data, where the F255A mutation significantly reduced IKACh block by chloroquine.Conclusion: Using numerical and structural biology approaches, we elucidated the details of how a small molecule could block an ion channel, and exert antiarrhythmic effects. Chloroquine binds the IKACh channel at a site formed by specific amino acids in the ion permeation pathway, leading to decreased IKACh, and the subsequent termination of AF.