Summary. Background: Peripheral serotonin (5-hydroxytryptamine, 5-HT) is transported by platelets and released upon stimulation. In the platelet cytoplasm, 5-HT is transamidated to small GTPases, promoting α-granule release and primary hemostasis. Objective: We hypothesized that 5-HT could also stimulate platelet receptor shedding after binding to the membrane 5-HT receptor (5-HT2AR). Methods: Western blot and flow cytometry were used to determine levels of the adhesion receptor glycoprotein (GP)Ibα on platelets or its shed fragment glycocalicin in plasma and serum from wild-type mice, Tph1−/− mice lacking peripheral 5-HT, and mice lacking functional tumor necrosis factor-alpha-converting enzyme (TACE, ADAM17). Flow chamber experiments and intravital microscopy were used to examine the adhesive properties of platelets after stimulation of 5-HT2AR. Results: Glycocalicin was significantly reduced in Tph1−/− plasma and serum. In isolated platelets, 5-HT induced shedding of GPIbα, which was increased to 60% when 5-HT uptake was inhibited by the selective serotonin reuptake inhibitor fluoxetine. Specific 5-HT2AR agonism and antagonism suggested activation of this receptor. The shedding could not be induced in TACEΔZn/ΔZn platelets, suggesting that activated TACE mediated the shedding of GPIbα. Intracellular signaling involved phosphorylation of p38 mitogen-activated protein kinase rather than G-protein signaling. 5-HT2AR stimulation decreased platelet adhesion to collagen-bound von Willebrand factor under arterial shear (1500 s−1) and incorporation into FeCl3-induced thrombi in mesenteric arterioles. Conclusions: Stimulation of 5-HT2AR on platelets induces TACE-mediated shedding of GPIbα, the key adhesion molecule under high shear conditions. Our observations demonstrate a new pathway through which 5-HT could modulate cardiovascular disease.