<p>Polyamines play essential roles in gene expression and modulate neuronal transmission in mammals. Vesicular polyamine transporters (VPAT) from the SLC18 family exploit the transmembrane H⁺ gradient to translocate polyamines into secretory vesicles, enabling the quantal release of polyamine neuromodulators and underpinning learning and memory formation. Here, we report the cryo-electron microscopy structures of human VPAT in complex with spermine, spermidine, H⁺, or tetrabenazine, elucidating discrete lumen-facing states of the antiporter and pivotal interactions between VPAT and its substrate or inhibitor. Leveraging structure-inspired mutagenesis studies and protein structure prediction, we deduce an unforeseen mechanism whereby the polyamine and H⁺ compete for multiple acidic protein residues both directly and indirectly, and rationalize how the antidopaminergic therapeutic tetrabenazine impedes vesicular transport of polyamines. This study unravels the mechanism of an H⁺-coupled polyamine antiporter, reveals mechanistic diversity between VPAT and other SLC18 antiporters, and raises new prospects for combating human disorders of polyamine homeostasis.</p>