Acetylcholine is a critical neuromodulator for aversive learning and cognitive memory. For its association with Alzheimer's disease, it has drawn much of attention as the therapeutic target. The therapeutic drug improves learning and memory deficits in Alzheimer's disease, but unable to cure. Muscarinic receptor M1 (M1R) has been implicated in aversive learning and cognitive memory through its downstream mediator protein kinase C (PKC) activation. However, the mode of action of acetylcholine in neurons has not been clarified yet, as PKC signaling pathway remains unknown. Based on that acetylcholine level is the highest in striatum/nucleus accumbens (NAc) where it impacts emotional behavior, in this study we aimed to clarify the M1R-PKC pathway in striatum/NAc leading to aversive learning. By exploiting our phosphoproteomics system for comprehensive PKC substrate screening, we found that acetylcholine activates Rho family GTPase Rac and its downstream effector PAK kinase. Also, we found that the activated Rac-PAK signaling by acetylcholine in striatopallidal medium spiny neuron contributes to aversive learning. This study would advance our understanding of the mode of action of Alzheimer's disease therapeutic drugs, shedding light to the molecular basis of Alzheimer's disease.