Imbalance of the autonomic nervous system is implicated in numerous cardiovascular pathologies. Enhanced cholinergic signalling in the heart has therapeutic potential and cholinesterase (ChE) inhibitors are under consideration for treating cardiovascular pathologies. Nevertheless, information about ChE in heart and their role in cardiac physiology are sparse. Here, we characterize the molecular composition, localization, and physiological functions of ChE in the mouse heart. Biochemical, microscopic, and physiological experiments were used to analyse ChE in mutant mice hearts lacking different ChE molecular forms.Both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were detected in the heart, with BChE activity exceeding AChE. The sinoatrial/atrioventricular nodes had the highest ChE activity with AChE being dominant. AChE was localized mainly in the atria and ventricular epicardium of the heart base. It was represented by two synaptic anchored forms (ColQ and PRiMA) and co-localized with the neuronal marker TUJ1. Mice with no functional AChE were unresponsive to ChE inhibition. Both detected anchored AChE participated in heart rate control. BChE was detected predominantly in ventricles, interestingly, as soluble monomers/dimers. Mice lacking BChE were more sensitive to ChE inhibition, confirming a scavenging role. It had a subtle impact on heart physiology, showing mainly a role in cholinergic antagonism to the positive inotropic effect of β-adrenergic stimulation. Our results suggest that AChE is part of the neuronal cholinergic system in the heart and participates in heart rate control; BChE is localized mainly to ventricular cardiomyocytes, scavenges ChE inhibitors, and participates in controlling the inotropic effect after adrenergic stimulation. Supported by APVV-22-0541.