During prolonged depolarization, voltage-gated K+ (Kv) channels display C-type inactivation, a process which is due to selectivity filter destabilization and serves to limit K+ flux. Here we reported that coumarsabin, a coumarin derivative isolated from Juniperus Sabina, could hasten C-type inactivation and thus cause block of Kv channels in neuronal NG108-15 cells and Kv1.2 channels heterologously expressed in lung epithelial H1355 cells. In NG108-15 cells, extracellular, but not intracellular, coumarsabin (30 μM) strongly speeded up Kv current decay and caused a left-shift in the steady-state inactivation curve. Coumarsabin inhibited end-of-pulse Kv currents with an IC50 of 13.4 μM. The kinetics and voltage-dependence of activation were not affected by coumarsabin. The degree of block by coumarsabin was not enhanced by a reduction in intracellular K+ concentration. Data reveal that coumarsabin was a closed channel blocker and it displayed a frequency-independent mode of inhibition. Coumarsabin did not accelerate current decay in a Kv1.2 mutant (V370G) defective in C-type inactivation. Taken together, our data suggest that Kv channel inhibition by coumarsabin did not appear to result from a direct obstruction of the outer pore but relied on C-type inactivation.