Significance Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential for the rhythmic activity of pacemaker cells in the heart and brain. How the movement of the voltage sensor, which is triggered by hyperpolarization of the membrane voltage, causes HCN channels to open is not clear. Here, we found that in HCN channels, the voltage sensor moves in two steps in response to hyperpolarizations and that the second voltage sensor step correlates with gate opening. A better understanding of HCN channel gating will aid in the development of better antiarrhythmic drugs.