Kv1.5 channels carry ultra-rapid delayed rectifier K+ currents in excitable cells, including neurons and cardiac myocytes. In the current study, the effects of cholinesterase inhibitor donepezil on cloned Kv1.5 channels expressed in HEK29 cells were explored using whole-cell recording technique. Exposure to donepezil resulted in a rapid and reversible block of Kv1.5 currents, with an IC50 value of 72.5 μM. The mutant R476V significantly reduced the binding affinity of donepezil to Kv1.5 channels, showing the target site in the outer mouth region. Donepezil produced a significant delay in the duration of activation and deactivation, and mutant R476V potentiated these effects without altering activation curves. In response to slowed deactivation time course, a typical crossover of Kv1.5 tail currents was clearly evident after bath application of donepezil. In addition, both this chemical and mutant R476V accelerated current decay during channel inactivation in a voltage-dependent way, but barely changed the inactivation and recovery curves. The presence of donepezil exhibited the use-dependent block of Kv1.5 currents in response to a series of depolarizing pulses. Our data indicate that donepezil can directly block Kv1.5 channels in its open and closed states.