The neurotransmitter ACh plays a crucial role in the communication between motor neuron and muscle at the neuromuscular junction (NMJ). Synthesis, storage and degradation of ACh are performed by choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and acetylcholinesterase (AChE), respectively. Presynaptic activity and nerve-induced muscle contraction regulate ACh release through muscarinic ACh autoreceptors (mAChRs). We previously identified a complex protein network including M1 and M2 mAChRs and multiple kinases that regulate ACh release at the NMJ. The aim of this work is to determine whether ChAT, VAChT and AChE protein levels are differentially modified by pre- and postsynaptic activities and whether M1 and M2 mAChRs are involved in this modulation. To study the effect of the synaptic activity on target proteins, the rat phrenic nerve was stimulated (1Hz, 30min) with or without contraction. Subsequently, pirenzepine and methoctramine, were used to block M1 and M2, respectively. Western Blot experiments showed that (1) presynaptic and postsynaptic activities don’t affect ChAT protein levels but accurately regulate VAChT and AChE; (2) M1 and M2 modulate ChAT and VAChT levels during presynaptic activity and nerve-induced muscle contraction. Super-resolution microscopy showed detailed location of ChAT and VAChT at the NMJ. In summary, the study provides evidence that M1 and M2 mAChRs regulate ChAT and VAChT protein levels during cholinergic neurotransmission, influencing acetylcholine availability at the NMJ. Thus, these findings open new perspectives on the regulation of cholinergic neurotransmission through the modulation of ChAT and VAChT by M1 and M2 mAChRs.*Equal-contribution-first-author.Funding:2021SGR01214,PID2019-106332GB-I00,PID2022-141252NB-I00,PRE2020-092084,2021-FI-B00755,2023PMF-PIPF-15.