Cytisine is a naturally occurring biomolecule, and, like its synthetic analogue varenicline, is a widely used and effective smoking cessation agent, an effect primarily modulated via nicotinic acetylcholine (nACh) receptors. Varenicline has a number of side effects, the most common of which is nausea and vomiting. This is likely caused by the activation of 5-HT₃ receptors, where varenicline has been shown to be a potent agonist. Cytisine has been less well studied, but has shown limited, if any, tendency to cause nausea and vomiting. Here we study the effects of cytisine on human 5-HT₃ receptors and compare them to the effects of varenicline. Displacement of the competitive 5-HT₃ receptor antagonist [³H]GR65630 reveals cytisine, like varenicline, binds to the 5-HT₃ receptor orthosteric binding site, but is 2000-fold less potent than varenicline, with an IC₅₀ of 0.5 mM. Functional studies show high cytisine concentrations partially inhibit 5-HT₃ receptor responses, in contrast to varenicline, which activates these proteins. A range of cytisine analogues show similar or lower potency than cytisine itself. As cytisine is a low potency antagonist of 5-HT₃ receptors, it would more likely prevent than cause the nausea and vomiting associated with 5-HT₃ receptor activation. Thus our data can explain why this side effect is not common for cytisine treatment, but is for varenicline treatment.