Persistent inward currents (PICs) play important roles in regulating neural excitability. Results from our previous studies showed that serotonergic (5-HT) neurons of the brainstem expressed PICs. However, little is known about cholinergic (ACh) modulation of PICs in the 5-HT neurons. The whole-cell patch-clamp recordings were performed in the brainstem slices of ePet-EYFP mice to investigate the electrophysiological properties of PICs with cholinergic modulation. PICs in 5-HT neurons were activated at - 51.4 ± 3.7 mV with the amplitude of - 171.6 ± 48.9 pA (n = 71). Bath application of 20-25 μM ACh increased the amplitude by 79.1 ± 42.5 pA (n = 23, p < 0.001) and hyperpolarized the onset voltage by 2.2 ± 2.7 mV (n = 23, p < 0.01) and half-maximal activation by 3.6 ± 2.7 mV (n = 6, p < 0.01). Muscarine mimicked the effects of ACh on PICs, while bath application of nicotine (15-20 μM) did not induce substantial change in the PICs (n = 9). Muscarine enhanced the amplitude of PICs by 100.0 ± 27.4 pA (n = 28, p < 0.001) and lowered the onset voltage by 2.8 ± 1.2 mV (n = 28, p < 0.001) and the half-maximal activation by 2.9 ± 1.4 mV. ACh-induced increase of amplitude and hyperpolarization of onset voltage were blocked by 3-5 μM atropine. Furthermore, the muscarine-induced enhancement of the PICs was antagonized by 5 μM 4-DAMP, the antagonist of M3 receptor, while the antagonists of M1 (Telenzepine, 5 μM) and M5 (VU6008667, 5 μM) receptors did not significantly affect the PIC enhancement. This study suggested that ACh potentiated PICs in 5-HT neurons of the brainstem by activating muscarinic M3 receptor.