Semaphorin 4D (Sema4D) is a transmembrane protein that supports contact-dependent amplification of platelet activation by collagen before being gradually cleaved by the metalloprotease ADAM17, as we have previously shown. Cleavage releases a soluble 120-kDa exodomain fragment for which receptors exist on platelets and endothelial cells. Here we have examined the mechanism that regulates Sema4D exodomain cleavage. The results show that the membrane-proximal cytoplasmic domain of Sema4D contains a binding site for calmodulin within the polybasic region Arg762-Lys779. Coprecipitation studies show that Sema4D and calmodulin are associated in resting platelets, forming a complex that dissociates upon platelet activation by the agonists that trigger Sema4D cleavage. Inhibiting calmodulin with W7 or introducing a membrane-permeable peptide corresponding to the calmodulin-binding site is sufficient to trigger the dissociation of Sema4D from calmodulin and initiate cleavage. Conversely, deletion of the calmodulin-binding site causes constitutive shedding of Sema4D. These results show that (1) Sema4D is a calmodulin-binding protein with a site of interaction in its membrane-proximal cytoplasmic domain, (2) platelet agonists cause dissociation of the calmodulin-Sema4D complex, and (3) dissociation of the complex is sufficient to trigger ADAM17-dependent cleavage of Sema4D, releasing a bioactive fragment.