<p>Human vesicular polyamine transporter (hVPAT) from the SLC18 antiporter family sequesters polyamine neuromodulators into secretory vesicles in exchange for H⁺ in the brain, thereby sustaining learning and memory formation. As a potential therapeutic target for combatting psychostimulant use disorder, hVPAT (or SLC18B1) regulates the homeostasis of monoamine neurotransmitters via an unknown mechanism. Here we report the cryo-electron microscopy structures of hVPAT in complexes with histamine and serotonin, revealing up to three distinct neurotransmitter-binding sites within the lumen-facing SLC18 antiporter. Complementary proteo-liposome-based transport and ligand-binding assays suggest that hVPAT transports multiple monoamine neurotransmitters simultaneously, exhibiting variable H⁺/substrate stoichiometry and positive cooperativity. Unexpectedly, this cooperativity appears to arise from the direct interactions between the bound histamine or serotonin molecules, which also contributes to substrate selectivity. In this work, our findings uncover a hitherto unidentified modality for monoamine neurotransmitter recognition and offer unprecedented insights into the general principles governing membrane transport.</p>