Abstract Background Cholinergic dysfunction occurs during aging and in a variety of diseases, including amyotrophic lateral sclerosis (ALS). However, it remains unknown whether changes in cholinergic transmission contributes to age- and disease-related degeneration of the motor system. Here we investigated the effect of moderately increasing levels of synaptic acetylcholine (ACh) on the neuromuscular junction (NMJ), muscle fibers, and motor neurons during development and aging and in a mouse model for amyotrophic lateral sclerosis (ALS). Methods Chat-ChR2-EYFP (VAChT Hyp ) mice containing multiple copies of the vesicular acetylcholine transporter (VAChT), mutant superoxide dismutase 1 (SOD1 G93A ), and Chat-IRES-Cre and tdTomato transgenic mice were used in this study. NMJs, muscle fibers, and α-motor neurons’ somata and their axons were examined using a light microscope. Transcripts for select genes in muscles and spinal cords were assessed using real-time quantitative PCR. Motor function tests were carried out using an inverted wire mesh and a rotarod. Electrophysiological recordings were collected to examine miniature endplate potentials (MEPP) in muscles. Results We show that VAChT is elevated in the spinal cord and at NMJs of VAChT Hyp mice. We also show that the amplitude of MEPPs is significantly higher in VAChT Hyp muscles, indicating that more ACh is loaded into synaptic vesicles and released into the synaptic cleft at NMJs of VAChT Hyp mice compared to control mice. While the development of NMJs was not affected in VAChT Hyp mice, NMJs prematurely acquired age-related structural alterations in adult VAChT Hyp mice. These structural changes at NMJs were accompanied by motor deficits in VAChT Hyp mice. However, cellular features of muscle fibers and levels of molecules with critical functions at the NMJ and in muscle fibers were largely unchanged in VAChT Hyp mice. In the SOD1 G93A mouse model for ALS, increasing synaptic ACh accelerated degeneration .