Organellar two-pore channels (TPCs) contain two copies of a Shaker-like six-transmembrane (6-TM) domain in each subunit and are ubiquitously expressed in plants and animals. Interestingly, plant and animal TPCs share high sequence similarity in the filter region yet exhibit drastically different ion selectivity. Plant TPC1 functions as a non-selective, cation channel on the vacuole membrane, while mammalian TPC channels have been shown to be endo/lysosomal Na+-selective or Ca2+-release channels. In this study, we performed systematic characterization of the ion selectivity of TPC1 from Arabidopsis thaliana (AtTPC1) and compared its selectivity to that of human TPC2 (HsTPC2). We demonstrate that AtTPC1 is slightly selective for Ca2+ over Na+, but non-selective among various group I monovalent cations. Our results also confirm that HsTPC2 is a highly Na+-selective channel activated by PI(3,5)P2. Guided by our recent structure of AtTPC1, we converted the non-selective AtTPC1 to a highly Na+-selective, HsTPC2-like channel and identified key residues in the TPC filters that differentiate the selectivity between AtTPC1 and HsTPC2. Furthermore, the structure of the Na+-selective AtTPC1 mutant elucidates the structural basis for Na+ selectivity in mammalian TPCs.