Acetylcholine (ACh) is an extensively studied neurotransmitter in animals. However, cholinergic mechanisms are not restricted to animals, but have also been reported in various plants. In the present study, we (1) characterized and quantified ACh and (2) evaluated the physiological impact of cholinergic receptors on growth and differentiation in the unicellular alga, Micrasterias denticulata. This species is one of the largest and most spectacularly shaped cells among the family Desmidiaceae. Therefore, it has been used as a model in studies on cell shape formation. Using high-performance liquid chromatography coupled with mass spectrometry, ACh was detected in Micrasterias. To quantify cholinergic effects on cell differentiation, we exposed young developmental stages of Micrasterias denticulata to different antagonists (d-tubocurarine, hexamethonium) and an agonist (nicotine). We found that cholinergic antagonists and, surprisingly, the agonist significantly suppressed cell growth and development. Thus, our results indicate that Micrasterias denticulata synthesize ACh and that ACh modulates growth and differentiation.