Arabidopsis thaliana two-pore channel AtTPC1 is a voltage-gated, Ca2+-modulated, non-selective cation channel that is localized in the vacuolar membrane and responsible for generating slow vacuolar (SV) current. Under depolarizing membrane potential, cytosolic Ca2+ activates AtTPC1 by binding at the EF hand domain whereas luminal Ca2+ inhibits the channel by stabilizing the voltage-sensing domain II (VSDII) in the resting state. Here we present 2.8-3.3 Å cryo-EM structures of AtTPC1 in two states, one in closed state with apo EF hand domain and resting VSDII, and the other in open or partially open state with Ca2+-bound EF hand domain and activated VSDII. Structural comparison between the two different states allows us to elucidate the structural mechanisms of voltage gating, cytosolic Ca2+ activation, and their coupling in AtTPC1. This study also provides structural insight into the general voltage-gating mechanism among voltage-gated ion channels.