Vagal nerve stimulation (VNS) holds a strong basis as a potentially effective treatment modality for chronic heart failure, which explains why a multicenter VNS study in heart failure with reduced ejection fraction is ongoing. However, more detailed information is required on the effect of acetylcholine (ACh) on repolarization in Purkinje and ventricular cardiac preparations to identify the advantages, risks, and underlying cellular mechanisms of VNS. Here, we studied the effect of ACh on the action potential (AP) of canine Purkinje fibers (PFs) and several human ventricular preparations. In addition, we characterized the effects of ACh on the L-type Ca²⁺ current (I_CaL) and AP of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and performed computer simulations to explain the observed effects. Using microelectrode recordings, we found a small but significant AP prolongation in canine PFs. In the human myocardium, ACh slightly prolonged the AP in the midmyocardium but resulted in minor AP shortening in subepicardial tissue. Perforated patch-clamp experiments on hiPSC-CMs demonstrated that 5 µM ACh caused an ≈15% decrease in I_CaL density without changes in gating properties. Using dynamic clamp, we found that under blocked K⁺ currents, 5 µM ACh resulted in an ≈23% decrease in AP duration at 90% of repolarization in hiPSC-CMs. Computer simulations using the O’Hara–Rudy human ventricular cell model revealed that the overall effect of ACh on AP duration is a tight interplay between the ACh-induced reduction in I_CaL and ACh-induced changes in K⁺ currents. In conclusion, ACh results in minor changes in AP repolarization and duration of canine PFs and human ventricular myocardium due to the concomitant inhibition of inward I_CaL and outward K⁺ currents, which limits changes in net repolarizing current and thus prevents major changes in AP repolarization.