Plants easily experience ammonia (NH4+) toxicity, especially aquatic plants. However, a unique wetland plant species, Myriophyllum aquaticum, can survive in livestock wastewater with more than 26 mM NH4+. In this study, the mechanisms of the M. aquaticum response to NH4+ toxicity were analysed with RNA-seq. Preliminary analysis of enzyme activities indicated that key enzymes involved in nitrogen metabolism were activated to assimilate toxic NH4+ into amino acids and proteins. In response to photosystem damage, M. aquaticum seemed to remobilize starch and cellulose for greater carbon and energy supplies to resist NH4+ toxicity. Antioxidative enzyme activity and the secondary metabolite content were significantly elevated for reactive oxygen species removal. Transcriptomic analyses also revealed that genes involved in diverse functions (e.g., nitrogen, carbon and secondary metabolisms) were highly responsive to NH4+ stress. These results suggested that a complex physiological and genetic regulatory network in M. aquaticum contributes to its NH4+ tolerance.