Proteolytic processing of the amyloid precursor protein (APP) by the beta- and gamma-secretases releases the amyloid-beta peptide (Abeta), which deposits in senile plaques and contributes to the etiology of Alzheimer's disease (AD). The alpha-secretase cleaves APP in the Abeta peptide sequence to generate soluble APPalpha (sAPPalpha). Upregulation of alpha-secretase activity through the 5-hydroxytryptamine 4 (5-HT4) receptor has been shown to reduce Abeta production, amyloid plaque load and to improve cognitive impairment in transgenic mouse models of AD. Consequently, activation of 5-HT4 receptors following agonist stimulation is considered to be a therapeutic strategy for AD treatment; however, the signaling cascade involved in 5-HT4 receptor-stimulated proteolysis of APP remains to be determined. Here we used chemical and siRNA inhibition to identify the proteins which mediate 5-HT4d receptor-stimulated alpha-secretase activity in the SH-SY5Y human neuronal cell line. We show that G protein and Src dependent activation of phospholipase C are required for alpha-secretase activity, while, unexpectedly, adenylyl cyclase and cAMP are not involved. Further elucidation of the signaling pathway indicates that inositol triphosphate phosphorylation and casein kinase 2 activation is also a prerequisite for alpha-secretase activity. Our findings provide a novel route to explore the treatment of AD through 5-HT4 receptor-induced alpha-secretase activation.