Purpose <p>Melatonin (MT) responds to various abiotic and biotic stresses. Herein, the effects of MT on alfalfa (<i>Medicago Sativa</i> L.) under high nitrate stress were studied.</p> Methods <p>Control (CK), 0.1 mmol L^− 1 MT (MT), 200 mmol L^− 1 nitrate (Nit), and 200 mmol L^− 1 nitrate + 0.1 mmol L^− 1 MT (Nit + MT) were conducted to investigate the response of alfalfa in morphology, tissue ultrastructure, photosynthesis, stomatal characteristics, ion balance, osmoregulation, cell membrane integrity, and redox homeostasis to high nitrate stress.</p> Results <p>Compared with Nit, Nit + MT significantly increased morphological parameters including plant height, leaf width, leaf area, taproot diameter, taproot length, fresh weight and dry weight; MT reduced the inhibitory effect of Nit on photosynthetic pigments and gas exchange parameters, protected leaf ultrastructure, increased chloroplast number, and reduced protoplast condensation and plasmolysis. MT significantly reduced total and open stomatal number, stomatal density, and open stomatal density under Nit stress. MT inhibited the NO₃¯ and NH₄⁺ toxicity, and maintained the metal ion balance. Nit reduced soluble sugar, proline, glycine betaine, and antioxidant system, relieving oxidative damage. Correlation analysis and principal component analysis revealed that most of the variables were significantly correlated, and Dim 1 (68.2%) and Dim 2 (7.5%) explained 75.7% of total variables.</p> Conclusions <p>MT can alleviate the ionic toxicity, osmotic stress and oxidative damage caused by high-nitrate stress, promote morphological growth, protect leaf tissues and pigment synthesis, and regulate stomatal state and redox homeostasis. Therefore, MT has potential application in reducing nitrogen pollution, promoting alfalfa growth and advancing sustainable agriculture.</p>