Vortioxetine (VTX) is a recently approved antidepressant that targets a variety of serotonin receptors. Here, we investigate the drug’s molecular mechanism of operation at the serotonin 5-HT₃ receptor (5-HT₃R), which features two properties: VTX acts differently on rodent and human 5-HT₃R, and VTX appears to suppress any subsequent response to agonists. Using a combination of cryo-EM, electrophysiology, voltage-clamp fluorometry and molecular dynamics, we show that VTX stabilizes a resting inhibited state of the mouse 5-HT₃R and an agonist-bound-like state of human 5-HT₃R, in line with the functional profile of the drug. We report four human 5-HT₃R structures and show that the human receptor transmembrane domain is intrinsically fragile. We also explain the lack of recovery after VTX administration via a membrane partition mechanism.