Acetylcholine (ACh) activates its nicotinic (nAChR) and muscarinic (mAChR) classes of receptors to modulate neuron excitability and cognitive functions dependent on the medial prefrontal cortex (mPFC). While the M1 excitatory mAChR is expressed on pyramidal neurons throughout the mPFC, recent data suggest that the M3 excitatory mAChR may be expressed selectively on pyramidal neurons within layer VI of the mPFC. Using whole-cell electrophysiological, we described through pharmacological and genetical manipulations the contribution of M3 mAChR to the excitability of layer VI pyramidal neurons within acute brain slices from adult mice of both sexes. Antagonism of M3 mAChR significantly decreased the overall muscarinic response, and its kinetics in both sexes. Transient local M3 mAChR downregulation also seems to have decreased the muscarinic response, such that a selective M3 mAChR antagonist would not have the same effect in the M3 mAChR knockdown group compared to controls. Our findings provide evidence of a layer-specific functional contribution of M3 mAChRs toward the role of ACh to support normal mPFC-dependent cognitive functions.