One hundred years after the discovery of acetylcholine (ACh) by Otto Lowei, ACh receptors, transporters and synthesizing and degrading enzymes became well-recognized contributors to cognition, neuromuscular, metabolic and immune processes. However, newer technologies identified unexpected molecular controllers over ACh signaling, including the SLEEPLESS, Isl1 and Lynx1 genes. These regulators are responsible, among other effects to the fine-tuned identity, functioning modes, dynamics and inter-cellular interactions of cholinergic cell types in and out of the brain, changing our understanding of ACh’s roles in human health and wellbeing. Furthermore, Genome-Wide Association Studies identify new disease-associated mutations and single nucleotide polymorphisms in coding and non-coding sequences within these genes. These discoveries add autism, amyotrophic lateral sclerosis, acute cardiac events, narcolepsy and obesity to the established acquired and inherited neuromuscular, stress-induced, dementia and epilepsy disorders that were traditionally associated with impaired ACh functioning. At the molecular level, cholinergic signaling involves both up- and down-regulation events of transcription, epigenetic modulations, alternative splicing and microRNA suppression that together coordinate the multi-targeted ACh signaling in brain and body functions and are also responsible to the reactions of patients to anti-cholinesterase therapeutics of Alzheimer’s disease as well as to global exposure to agricultural pesticides and to individual tendencies for nicotine addiction, calling for new basic and translational research venues for regulating ACh signaling. Integrating these molecular ACh regulators into every discussion of cholinergic issues, should incorporate data obtained by clinicians and molecular geneticists, neuroscientists and structural biochemists over the past decades into a refreshed look at the intricate checks and balances over cholinergic signaling. Our understanding of the cholinergic regulators is incomplete, but time is ripe to summarize the recent reports on checks and balances of cholinergic signaling and their implications in health and disease.