Abstarct: Salinity stress is one of the most important abiotic stresses that affects crop yields and is currently becoming one of the most important limiting factors for crop cultivation in the world. Identification of physiological, biochemical and molecular responses of plants to salinity stress is effective in increasing human knowledge and identifying the mechanisms of plants to salinity stress. The aim of this study was to evaluate the response of fenugreek plants treated with different levels of melatonin under salinity stress. Also in this study, the effects of melatonin as a functional stimulant on diosgenin content were investigated. The results of this study showed that the use of different levels of melatonin effectively prevents the degradation of plant pigments. In addition, melatonin increased the content of antioxidants by stimulating genes involved in the biosynthetic pathway, thereby reducing the content of active oxygen species and free radicals. The result was a reduction in free radicals, a decrease in electrolyte leakage and malondialdehyde content, and finally an increase in the resistance of fenugreek plants under salinity stress. In addition, the application of different levels of melatonin by increasing the activity of potassium-carrying channels led to the maintenance of ionic homeostasis and increased intracellular water content under salinity stress. In this study, the interaction effects of melatonin, auxin and abscisic acid in fenugreek treated with different salinity levels were investigated for the first time. The results of this study showed that melatonin through the pathways dependent on abscisic acid and nitric acid leads to the activation of defense signaling pathways in fenugreek. Our findings showed that melatonin effectively increases the exxpression of genes involved in the biosynthesis pathway of diosgenin and consequently the diosgenin content. In fact, the combined combination of 150 mM salinity stress and 60 ppm melatonin increased the diosgenin content by more than six times compared to the normal condition. The results of this study increased our knowledge about the melatonin effects on fenugreek under salinity stress and also on changes in the diosgenin content in fenugreek. Finally, our findings demonstrate that melatonin as an important functional stimulant is able to induce physiological, biochemical (primary and secondary mextabolites) and molecular changes in salinity-treated fenugreek in order to increase their resistance.