This study presents cryo-EM structures of human vesicular monoamine transporter 2 (VMAT2) in three distinct states: apo (without ligand), substrate-bound (serotonin), and inhibitor-bound (tetrabenazine and reserpine). These structures reveal the structural basis of VMAT2's substrate recognition and its inhibition by various inhibitors. The apo state is lumen-facing, while the inhibitor-bound states are occluded and cytosol-facing. Tetrabenazine induces a unique conformational change involving TM2 and TM7, extending beyond the typical rocker-switch movement. The structures, combined with biochemical analysis, elucidate the proton-driven exchange cycle and provide insights into the design of improved VMAT2-targeting drugs. VMAT2 is crucial for the storage and release of monoamine neurotransmitters such as serotonin, dopamine, and norepinephrine in neurons. It is a valid pharmaceutical target for treating various neurological and psychiatric disorders. The study shows that VMAT2 has a higher affinity for catecholamines compared to serotonin, and non-competitive inhibitors like tetrabenazine selectively target VMAT2. The structures reveal the binding sites for substrates and inhibitors, as well as the conformational changes that occur during transport. The study also highlights the structural differences between VMAT2 and its paralog VMAT1, and the distinct pharmacological effects of competitive and non-competitive inhibitors. The cryo-EM structures provide a framework for understanding the transport mechanism of VMAT2, including the role of proton gradients and the conformational changes that facilitate substrate transport. The findings contribute to the development of more effective drugs targeting VMAT2 for the treatment of neuropsychiatric diseases.This study presents cryo-EM structures of human vesicular monoamine transporter 2 (VMAT2) in three distinct states: apo (without ligand), substrate-bound (serotonin), and inhibitor-bound (tetrabenazine and reserpine). These structures reveal the structural basis of VMAT2's substrate recognition and its inhibition by various inhibitors. The apo state is lumen-facing, while the inhibitor-bound states are occluded and cytosol-facing. Tetrabenazine induces a unique conformational change involving TM2 and TM7, extending beyond the typical rocker-switch movement. The structures, combined with biochemical analysis, elucidate the proton-driven exchange cycle and provide insights into the design of improved VMAT2-targeting drugs. VMAT2 is crucial for the storage and release of monoamine neurotransmitters such as serotonin, dopamine, and norepinephrine in neurons. It is a valid pharmaceutical target for treating various neurological and psychiatric disorders. The study shows that VMAT2 has a higher affinity for catecholamines compared to serotonin, and non-competitive inhibitors like tetrabenazine selectively target VMAT2. The structures reveal the binding sites for substrates and inhibitors, as well as the conformational changes that occur during transport. The study also highlights the structural differences between VMAT2 and its paralog VMAT1, and the distinct pharmacological effects of competitive and non-competitive inhibitors. The cryo-EM structures provide a framework for understanding the transport mechanism of VMAT2, including the role of proton gradients and the conformational changes that facilitate substrate transport. The findings contribute to the development of more effective drugs targeting VMAT2 for the treatment of neuropsychiatric diseases.