Mesenchymal stem cells (MSCs) are under the control of a large number of signaling systems. In this study, the presence and functionality of the acetylcholine (ACh) signaling system in MSCs was examined. We detected the expression of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and the presence of ACh in MSCs. MSCs also expressed the nicotinic acetylcholine receptor subunits α3, α5, α7, and the muscarinic acetylcholine receptor 2 (M2-receptor). The M2-receptor and the nicotinic α7 receptor subunits were expressed on distinct subpopulations of cells, indicating differential regulation of cholinergic signaling between MSCs. Stimulation of MSCs with the nicotinic receptor agonist nicotine and the muscarinic receptor agonist muscarine induced immediate and transient increases in intracellular Ca2+concentration. Furthermore, muscarine had an inhibiting effect on the production of the intracellular signaling molecule cyclic adenosine 3′,5′-monophosphate (cAMP). The AChE inhibitor chlorpyrifos, which is widely used as an agricultural insecticide, had similar effects on intracellular Ca2+and cAMP in MSCs. Nicotine, muscarine, and chlorpyrifos induced the phosphorylation of extracellular signal-regulated kinases 1 and 2. This study demonstrates that several components of a cholinergic signaling system are present and functional in MSCs. Environmental compounds such as nicotine and agricultural insecticides can interfere with this system and may affect cellular processes in the MSC.