Acetylcholinesterase (AChE) plays an essential role in neuromuscular transmission, therefore it is surprising that AChE knockout (KO) mice could live to the adulthood. Neuromuscular functioning in KO and normal (wild type, WT) mice were studied, at different age (1.5-, 4- and 9-month-old). Hindlimb muscle force productions in response to nerve or muscle electric stimulation were recorded in situ and in vitro. Our results show that contrary to WT mice, 1.5-, 4- and 9-month-old KO mice exhibited a decreased in tetanic force during short periods (500 ms) of repetitive nerve stimulations (tetanic fade). Nevertheless submaximal muscle forces in response to single or repetitive nerve stimulation were increased (potentiation) in 1.5-, 4- and 9-month-old KO mice as compared to WT mice (p < 0.05). Tetanic fade and potentiation were absent when muscles were directly stimulated, indicating neuromuscular transmission alterations in KO mice. Contrary to younger mice, muscle weight and maximal tetanic force in response to repetitive nerve stimulation were not reduced in 4- and 9-month-old KO mice as compared to WT mice (p > 0.05). In conclusion AChE deficit leads to marked neuromuscular alterations in hind limb muscle functioning and a prominent symptom is the lack of resistance to fatigue.