Mesenchymal stem cells (MSCs) are well known for their regenerative potential. Even though the ability of MSCs to proliferate and differentiate has been studied extensively, there remains much to learn about the signaling mechanisms and pathways that control proliferation and influence the differentiation phenotype. In recent years, there has been growing evidence for the utility of non-neuronal cholinergic signaling systems and that acetylcholine (ACh) plays an important ubiquitous role in cell-to-cell communication. Indeed, cholinergic signaling is hypothesized to occur in stem cells and ACh synthesis, as well as in ACh receptor (AChR) expression, has been identified in several stem cell populations, including MSCs. Furthermore, AChRs have been found to influence MSC regenerative potential. In humans, there are two major classes of AChRs, muscarinic AChRs and nicotinic AChRs, with each class possessing several subtypes or subunits. In this review, the expression and function of AChRs in different types of MSC are summarized with the aim of highlighting how AChRs play a pivotal role in regulating MSC regenerative function.