The $G_{q/11}$ protein-coupled receptors, such as muscarinic (M1 & M3) receptors, have been shown to regulate the release of a soluble amyloid precursor protein (sAPP$alpha$) produced from $alpha$-secretase processing. However, there is no direct evidence for the precise characteristics of G proteins, and the signaling mechanism for the regulation of $G_{q/11}$ protein-coupled receptor mediated sAPP$alpha$ release is not clearly understood. This study examined whether the muscarinic receptor-mediated release of sAPP$alpha$ is directly regulated by $Galpha_{q/11}$ proteins. The HEK293 cells were transiently cotransfected with muscarinic M3 receptors and a dominant-negative minigene construct of the G protein $alpha$ subunit. The sAPP$alpha$ release in the media was measured using an antibody specific for sAPP. The sAPP$alpha$ release enhancement induced by muscarinic receptor stimulation was decreased by a $G_{q/11}$ minigene construct, whereas it was not blocked by a control minigene construct (the G$alpha$ carboxy peptide in random order, G$alpha_{q}$R) or $Galpha_{j}$ constructs. This indicated a direct role of the $Galpha_{q/11}$ protein in the regulation of muscarinic M3 receptor-mediated sAPP$alpha$ release. We also investigated whether the transactivation of the epidermal growth factor receptor (EGFR) by a muscarinic agonist could regulate the sAPP$alpha$ release in SH-SY5Y cells. Pretreatment of a specific EGFR kinase inhibitor, tyrophostin AG1478 (250 nM), blocked the EGF-stimulated sAPP$alpha$ release, but did not block the oxoM­stimulated sAPP$alpha$ release. This demonstrated that the transactivation of the EGFR by muscarinic receptor activation was not involved in the muscarinic receptor-mediated sAPP$alpha$ release.