Acetylcholinesterase (AChE) is a primary regulator of cholinergic signaling within and out of the central nervous system. It has been studied extensively for several decades, yet many basic questions about its regulation, composition and functions remain open. Thus, neither the full impact of AChE’s activities on neuronal development, maintenance and functioning nor the reasons for why and how different AChE molecular forms are being synthesized and degraded are fully known yet, although recent evidence suggests the involvement of micro-RNA regulators in both of these aspects. The identification of the anchoring proteins, PRiMA and ColQ, stimulates studies on AChE’s assembly and its specific localization in the brain and muscle. Additionally, non-cholinergic function(s) of AChE has been proposed as relevant both for neuronal differentiation and cognitive functioning, whereas transgenic engineering, recombinant production and structure-function analyses of this enzyme’s variants with various inhibitors paved new avenues for the development of novel Alzheimer’s disease therapeutics, agricultural pesticides and prophylactics in anticipation of warfare agent exposure. The aims of this Research Topic is to provide a forum for experts in the field to critically discuss recent developments in AChE studies, initiate global critiques of the current views for resolving the fundamental questions in AChE research and promote the development of novel versions and uses of AChE-targeted agents. Topics of interest include but are not limited to: (i) post-transcriptional regulation of AChE; (ii) the non-cholinergic functions of AChE; and (iii) anti-AChE inhibitors and diseases.