Amsacrine is an antineoplastic agent for acute lymphoblastic leukemia. It contains methanesulfonamide group —similar to hERG/I _Kr inhibitors like E-4031 and dofetilide—therefore may exert cardiac arrhythmias. Although amsacrine inhibits expressed hERG channels, its effects on native cardiac electrophysiology remain unclear. We investigated the effects of 1 and 10 µM amsacrine on action potentials (APs) and underlying ionic currents in isolated canine ventricular cardiomyocytes. Amsacrine reduced phase 0 parameters: maximal upstroke velocity (∼50–60%), action potential amplitude (∼15%), and peak potential (∼55%), suggesting inhibition of sodium current. Moreover, the maximal rates of phase 1 and 3 repolarizations, and plateau potentials were reduced, suggesting effects on multiple ionic currents. Despite these, AP duration (APD ₂₀ –APD ₉₀ ) remained unaffected. AP voltage-clamp analysis revealed a complex amsacrine-sensitive current consisting of an early inward component consistent with sodium current inhibition, a second early outward component suggesting transient outward current suppression, a mid-plateau inward component likely reflecting inhibition of inward plateau currents, and a late outward component compatible with I _Kr inhibition. 10 µM amsacrine reduced L -type calcium, transient outward potassium and expressed Na _V 1.5 channel currents by 22, 38, and 10%, respectively. Native I _Kr was also reduced in a concentration-dependent manner (152 nM IC ₅₀ and 1.21 Hill coefficient). These findings demonstrate that amsacrine modulates multiple cardiac ion currents. The lack of AP prolongation despite I _Kr inhibition can be due to concomitant suppression of inward currents. As the tested concentrations overlap with therapeutic plasma levels (3–18 µM), amsacrine may carry a non-negligible risk of cardiac electrophysiological effects, particularly in susceptible patients.