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Background Humans are exposed to nitrate predominantly through diet with peak plasma concentrations within an hour after ingestion, but additional exposure is obtained from the environment, and minimally through de novo synthesis. Higher nitrate consumption has been associated with methemoglobinemia, spontaneous abortions, atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, amyotrophic lateral sclerosis, and neural tube defects. However, skeletal muscle development has not been examined. Methods C2C12 skeletal muscle cell cultures were maintained, myoblasts were fused into myotubes, and then cultures were exposed to motor neuron derived agrin to enhance acetylcholine receptor (AChR) clustering. Untreated cultures were compared with cultures exposed to sodium nitrate at concentrations ranging from 10 ng/mL–100 μg/mL. Results The results reported here demonstrate that 1 μg/mL sodium nitrate was sufficient to decrease the frequency of agrin-induced AChR clustering without affecting myotube formation. In addition, concentrations of sodium nitrate of 1 μg/mL or 100 μg/mL decreased gene expression of the myogenic transcription factor myogenin and AChR in correlation with the agrin-induced AChR clustering data. Conclusions These results reveal that sodium nitrate decreases the frequency of agrin-induced AChR clustering by a mechanism that includes myogenin and AChR gene expression. As a consequence sodium nitrate may pose a risk for skeletal muscle development and subsequent neuromuscular synapse formation in humans.