Aconitine (ACO), a highly toxic diterpenoid alkaloid, is recognized to have effects on cardiac voltage–gated Na+ channels. However, it remains unknown whether it has any effects on K+ currents. The effects of ACO on ion currents in differentiated clonal cardiac (H9c2) cells and in cultured neonatal rat ventricular myocytes were investigated in this study. In H9c2 cells, ACO suppressed ultrarapid-delayed rectifier K+ current (IKur) in a time- and concentration-dependent fashion. The IC50 value for ACO-induced inhibition of IKur was 1.4μM. ACO could accelerate the inactivation of IKur with no change in the activation time constant of this current. Steady-state inactivation curve of IKur during exposure to ACO could be demonstrated. Recovery from block by ACO was fitted by a single-exponential function. The inhibition of IKur by ACO could still be observed in H9c2 cells preincubated with ruthenium red (30μM). Intracellular dialysis with ACO (30μM) had no effects on IKur. IKur elicited by simulated action potential (AP) waveforms was sensitive to block by ACO. Single-cell Ca2+ imaging revealed that ACO (10μM) alone did not affect intracellular Ca2+ in H9c2 cells. In cultured neonatal rat ventricular myocytes, ACO also blocked IKur and prolonged AP along with appearance of early afterdepolarizations. Multielectrode recordings on neonatal rat ventricular tissues also suggested that ACO-induced electrocardiographic changes could be associated with inhibition of IKur. This study provides the evidence that ACO can produce a depressant action on IKur in cardiac myocytes.