Tanshinone IIA, one of the main active components from the Chinese herb Danshen, is widely used to treat cardiovascular diseases in Asian countries, especially in China. To further elucidate its heart rate–reducing and anti-ischemic mechanisms, here we investigated the effects of tanshinone IIA on hyperpolarization-activated cyclic nucleotide–modulated (HCN) channels expressed in Xenopus oocytes using two-electrode voltage clamp techniques. When applied to the extracellular solution, 100 μM tanshinone IIA caused a slowing of activation and deactivation and an increase of minimum open probabilities (from 0.06 ± 0.01 to 0.29 ± 0.03, P<0.05) in HCN2 channels without shifting the voltage dependence of channel activation. Tanshinone IIA potently enhanced the amplitude of voltage-independent current (instantaneous current) of HCN2 at −90 mV in a concentration-dependent manner with an EC₅₀ of 107 μM. Similar but 2.3-fold less sensitivity to tanshinone IIA was observed in the HCN1 subtype. More significant effect on HCN2 and MiRP1 co-expression was observed. In conclusion, tanshinone IIA changed HCN channel gating by selectively enhancing the instantaneous current (one population of HCN channels), which resulted in the corresponding increment of minimum open probabilities, slowing channel activation and deactivation processes with little effect on the voltage-dependent current (another population of HCN channels).