Brain function is one of the most distinctive and important aspects of human physiology. Chemical messengers, known as neurotransmitters, are involved in the conduction of signals by the nervous system. In 1921, Otto Löwi discovered that a chemical transmitter which he called ‘Vagusstoff’, released from vagal nerve terminals, decelerated the rate of heartbeats. This was later shown to be the action of acetylcholine. Acetylcholine is a major neurotransmitter of the central, as well as the peripheral, nervous system. The human body has specific receptors for acetylcholine (i.e., cholinergic receptors) through which acetylcholine produces a wide range of physiological effects. These physiological effects play an important role in maintaining homeostasis in the human body. Alteration in the acetylcholine levels or modified receptor expression and function in specific parts of the nervous system are known to be responsible for various neurodegenerative diseases. Recently, apart from these traditional neurodegenerative diseases, the role of the extraneuronal cholinergic system has come to light in several serious diseases, including cancer, hypotension, and even diabetes. Understanding the basic principles of cholinergic neurotransmission is the first step in leveraging this system to combat such diseases. Hence, this chapter attempts to explain the working of the cholinergic system and its role in the pathophysiology of diseases. © 2023 Taylor & Francis Group, LLC.