Increasing soil salinity suppresses both productivity and fiber quality of cotton, thus, an appropriate management approach needs to be developed to lessen the detrimental effect of salinity stress. This study assessed two cotton genotypes with different salt sensitivities to investigate the possible role of nitrogen supplementation at the seedling stage. Salt stress induced by sodium chloride (NaCl, 200 mmol·L −1 ) decreased the growth traits and dry mass production of both genotypes. Nitrogen supplementation increased the plant water status, photosynthetic pigment synthesis, and gas exchange attributes. Addition of nitrogen to the saline media significantly decreased the generation of lethal oxidative stress biomarkers such as hydrogen peroxide, lipid peroxidation, and electrolyte leakage ratio. The activity of the antioxidant defense system was upregulated in both saline and non-saline growth media as a result of nitrogen application. Furthermore, nitrogen supplementation enhanced the accumulation of osmolytes, such as soluble sugars, soluble proteins, and free amino acids. This established the beneficial role of nitrogen by retaining additional osmolality to uphold the relative water content and protect the photosynthetic apparatus, particularly in the salt-sensitive genotype. In summary, nitrogen application may represent a potential strategy to overcome the salinity-mediated impairment of cotton to some extent.Plants 2020, 9, 450 2 of 20 most sensitivity to salinity during the seedling growth stage, and remained sensitive at the reproductive stage [9]. Excess salinity inhibits plant growth and development by inducing osmotic stress, particular ion toxicity (Na + and Cl − ), oxidative damage, and/or nutritional disorders in plant tissues, which subsequently lead to weakened plant growth and endurance [10][11][12]. Salt stress has been shown to significantly decrease the uptake and metabolism of numerous mineral nutrients (such as nitrogen, potassium, phosphorus, and calcium), and accelerate the damage of the photosynthetic machinery as well as the whole salt tolerance mechanisms of plants. Salt stress-induced plant metabolism alterations are the secondary consequence of carbon and nitrogen metabolism [13]. Moreover, a high salt concentration perturbs electron transport systems in both chloroplasts and mitochondria [14,15]. This accelerates electron leakage from electron transportation chains and induces the generation of reactive oxygen species (ROS), such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide [16,17]. The over-accumulation of ROS not only damages cellular and biological activities [18][19][20], but also degrades chlorophyll pigments and obstructs the synthesis of proteins, amino acids, lipids, deoxyribonucleic acid, as well as enzymatic and non-enzymatic activities of plants [17]. To avoid ROS-induced oxidative damage, plant-assured endogenous tolerance approaches, such as the antioxidant defense system as well as the accumulation of organic solutes and secondary metabolites [21]....