Acetylcholine esterases (AChEs) are essential enzymes in cholinergic synapses, terminating neurotransmission by hydrolyzing acetylcholine. Membrane bound AChEs at synaptic clefts efficiently perform this task, while soluble AChEs are less stable and effective, but function over broader areas. Vertebrates typically have one AChE gene producing alternatively spliced forms, whereas some invertebrates have multiple AChE genes, producing both synaptic and soluble enzymes. Despite extensive research of invertebrate AChEs as pesticide targets, their physiological roles remain largely elusive. We characterized seven AChEs in the wandering spider, Cupiennius salei, a model species for developmental and neurophysiological studies. Structural and phylogenetic analyses identified CsAChE7 as the sole stable, membrane-bound enzyme likely functioning at synaptic clefts, while the other six are soluble enzymes. In situ hybridization revealed CsAChE7 expression in the nervous system, particularly in cells also expressing choline acetyltransferase and exhibiting AChE activity. In the periphery, CsAChE7 was found in a subgroup of rapidly adapting mechanosensory neurons, facilitating precise and transient activation of postsynaptic cells. Conversely, slowly adapting neurons, also cholinergic, within the same sensory organ, express only soluble AChEs, leading to prolonged postsynaptic activation. Therefore, cholinergic transmission is not only dictated by postsynaptic receptors but also by the characteristics of the enzymes clearing acetylcholine from the synapse. We also show that acetylcholine is a crucial neurotransmitter in the spider’s visual system, sensory and motor pathways, but absent in excitatory motor neurons at neuromuscular junctions, consistent with other arthropods. Our findings on sequence structures may significantly impact the development of neurological drugs and pesticides.