Background Selective inhibitors of K_v1.5 channels are being developed for the treatment of atrial fibrillation (AF). Objectives The purpose of this study was to investigate the effects of the highly selective K_v1.5 inhibitor XEN-D0103 on human atrial action potentials (APs) at high excitation rates and to assess safety. Methods Intracellular APs (stimulation rates 1-5 Hz) were measured in right atrial trabeculae from patients in sinus rhythm (SR), chronic AF (cAF; AF of >6 months duration), and paroxysmal AF (pAF). The safety and tolerability of XEN-D0103 were tested in a double-blind, randomized, placebo-controlled phase 1 study. Results Depending on its concentration, XEN-D0103 elevated the plateau potential. At 1 Hz, XEN-D0103 (3 μM) shortened action potential duration at 90% repolarization (APD₉₀) and effective refractory period (ERP) in SR preparations, but prolonged these parameters in cAF preparations. In SR and pAF preparations, the shortening effects on APD₉₀ and ERP turned into prolongation at high rates. In cAF trabeculae, XEN-D0103 prolonged APD₉₀ and ERP at 2 and 3 Hz. At high rates, more SR and pAF preparations failed to capture excitation in the presence of the drug than in its absence. XEN-D0103 (10 μM) did not significantly affect human ventricular APs. Even with plasma concentrations reaching 7000 ng/mL, XEN-D0103 did not increase δδQTcF (QT interval corrected by the Fridericia formula) in the analysis of electrocardiograms of healthy volunteers, and no subjects receiving an active treatment had a QT or QTcF interval >450 ms, or increase in QTcF from baseline >30 ms. Conclusion APD prolongation and suppression of APs by XEN-D0103 at high stimulation rates in SR and pAF tissue, but not cAF, could be of therapeutic benefit for reducing AF burden. This concept needs to be confirmed in clinical trials.