© 2017 The effect of water-soluble pristine C60 fullerene nanoparticles (C60NPs) on receptor-operated cation channels formed by TRPC4/C6 proteins in ileal smooth muscle cells was investigated for the first time. Activation of these channels subsequent to acetylcholine binding to the expressed in these cells M2 and M3 muscarinic receptors represents the key event in the parasympathetic control of gastrointestinal smooth muscle motility and cholinergic excitation-contraction coupling. Experiments were performed on single collagenase-dispersed mouse ileal myocytes using patch-clamp techniques with symmetrical 125 mM Cs+ solutions and [Ca2 +]i ‘clamped’ at 100 nM in order to isolate the muscarinic cation current (mICAT). The current was induced by intracellular infusion of 200 μM GTPγS, which activates G-proteins directly, i.e. bypassing the muscarinic receptors. C60NPs applied at 10− 6 M at peak response to activation of G-proteins caused mICAT inhibition by 47.0 ± 3.5% (n = 9). The inhibition developed rather slowly, with the time constant of 119 ± 16 s, was voltage-independent and irreversible. Thus, C60NPs are unlikely to cause any direct block of TRPC4/C6 channels; rather, they may accumulate in the membrane and disrupt G-protein signalling leading to mICAT generation. C60NPs may represent a novel class of biocompatible molecules for the treatment of disorders associated with enhanced gastrointestinal motility.