Two-pore channels, or two-pore Na+ channels (TPCs), contain two homologous domains, each containing a functional unit typical of voltage-dependent cation channels. Each domain is considered to be responsible for either phosphoinositide (PI) binding or voltage sensing. Among the three members of the TPC family, TPC1 and TPC2 are activated by PI(3,5)P2, while TPC3 has been thought not to be affected by any PIs. Here, we report that TPC3 is sensitive to PI(3,4)P2 and PI(3,5)P2, but not to PI(4,5)P2, and that the extremely slow increase in TPC3 currents induced by depolarization in Xenopus oocytes is due to the production of PI(3,4)P2. Similarly to TPC1, the cluster of basic amino acid residues in domain I is critical for PI sensitivity, but with a slight variation that may allow TPC3 to be sensitive to both PI(3,4)P2 and PI(3,5)P2. We also found that TPC3 has a unique PI-dependent modulation mechanism of voltage dependence, which is achieved by a specific bridging interaction between domain I and domain II. Taken together, these findings show that TPC3 is a unique member of the TPC family that senses PIs and displays a strong coupling between PI binding and voltage-dependent gating.