The anthelmintic drug praziquantel (PZQ) causes contraction of parasitic schistosomes, as well as constriction of blood vessels within the mesenteric vasculature of the host where the adult blood flukes reside. The contractile action of PZQ on the vasculature is mediated by activation of host serotonergic 5-HT2B receptors. However, and the molecular basis for PZQ interaction with these targets and the location of these 5-HT2B receptors in the vessel wall has not been experimentally defined. Evaluation of a PZQ docking pose within the 5-HT2BR orthosteric site, using both Ca2+ reporter and bioluminescence resonance energy transfer (BRET) assays, identified residues F340 and F341 (transmembrane helix 6, TM6) as well as L209 (extracellular loop 2) as critical for PZQ-mediated agonist activity. A key determinant of PZQ selectivity for the 5-HT2B receptor over the 5-HT2A/2C receptors was determined by M2185.39 in transmembrane helix 5 (TM5) of the orthosteric site. Mutation of this residue to valine (M218V), as found in 5-HT2A and 5-HT2C, decreased PZQ agonist activity, whereas the reciprocal mutation (V215M) in 5-HT2C increased PZQ activity. Two-photon imaging in intact mesenteric arterial strips visualized PZQ-evoked Ca2+ transients within the smooth muscle cells of the vessel wall. PZQ also triggered cytoplasmic Ca2+ signals in arterial smooth muscle cells in primary culture that were isolated from mesenteric blood vessels. These data define the molecular basis for PZQ action on 5-HT2B receptors localized in vascular smooth muscle.