There is scarce information on the fate of cardiac progenitor cells (CPC) in the embryonic heart after chamber specification. Furthermore, the role of acetylcholinesterase (AChE) during heart development is unknown, despite record of its presence in the myocardium. Although three molecular variants of AChE (R, H and T) exist due to alternate splicing, temporal and spatial distribution of these splice variants during cardiac ontogeny is not well characterized. We hypothesized that the AChE “R” splice variant (AChE-R) is involved in directing lineage commitment of mouse ventricular CPCs to the conduction cell phenotype. It is possible that AChE may promote the breakdown of ACh and block the effects of ligand-binding via M2 receptors present on the surface of CPCs. Our study has also provided a platform to suggest that AChE may play a role in the molecular mechanisms underlying functional diversification of myocardial cells into conduction system cells during ontogenesis.