The muscarinic acetylcholine receptor (mAChR) has been considered one of the neurotransmitter receptors regulating hippocampal synaptic plasticity, which likely plays a critical role in learning and memory. In previous studies, however, muscarinic agonists were used at relatively high concentrations, and the subtype selectivity of muscarinic antagonists was not satisfactory. Thus, it remains to be answered whether physiological levels of ACh are involved in the regulation of synaptic plasticity and which mAChR subtypes are responsible for such effects. We found in this study that a low concentration (50 nm) of carbachol enhanced long-term potentiation (LTP) of excitatory synaptic transmission in mouse hippocampal slices. Notably, this enhancing effect was abolished in M1 mAChR knock-out (KO) but not in M3 mAChR KO mice, although LTP itself was intact in both mutant mice. Furthermore, we found that repetitive stimulation in the stratum oriens, which presumably triggered the release of endogenous ACh from cholinergic terminals, could enhance LTP in wild-type mice but not in M1 mAChR KO mice. These results suggest that physiologically released ACh from cholinergic fibers modulates hippocampal synaptic plasticity through the postsynaptic M1 mAChR activation.