Acetylcholine can excite neurons by suppressing M-type (KCNQ) potassium channels. This effect is mediated by M1muscarinic receptors coupled to the Gqprotein. Although PIP2depletion and PKC activation have been strongly suggested to contribute to muscarinic inhibition of M currents (IM), direct evidence is lacking. We investigated the mechanism involved in muscarinic inhibition of IMwith Ca2+measurement and electrophysiological studies in both neuronal (rat sympathetic neurons) and heterologous (HEK cells expressing KCNQ2/KCNQ3) preparations. We found that muscarinic inhibition of IMwas not blocked either by PIP2or by calphostin C, a PKC inhibitor. We then examined whether muscarinic inhibition of IMuses multiple signaling pathways by blocking both PIP2depletion and PKC activation. This maneuver, however, did not block muscarinic inhibition of IM. Additionally, muscarinic inhibition of IMwas not prevented either by sequestering of G-protein βγ subunits from Gα-transducin or anti-Gβγantibody or by preventing intracellular trafficking of channel proteins with blebbistatin, a class-II myosin inhibitor. Finally, we re-examined the role of Ca2+signals in muscarinic inhibition of IM. Ca2+measurements showed that muscarinic stimulation increased intracellular Ca2+and was comparable to the Ca2+mobilizing effect of bradykinin. Accordingly, 20-mM of BAPTA significantly suppressed muscarinic inhibition of IM. In contrast, muscarinic inhibition of IMwas completely insensitive to 20-mM EGTA. Taken together, these data suggest a role of Ca2+signaling in muscarinic modulation of IM. The differential effects of EGTA and BAPTA imply that Ca2+microdomains or spatially local Ca2+signals contribute to inhibition of IM.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10571-022-01303-7.