Abstract: Chloride stress is one of the main factors limiting the yield and quality of watermelon in Ningxia due to long-term irrigation with underground chlorinated brackish water. Nitrogen could alleviate the chlorine toxicity of crops. Therefore, it is crucial to explore the regulatory mechanism of nitrogen on the chloride stress by applying nitrogen fertilizer rationally and controlling chlorosis of watermelon under chloride stress. In this study, a soil culture experiment was conducted to determine the effects of different nitrogen application rates 0, 0.10, 0.15, 0.20, 0.25 g∙kg⁻¹ (oven-dry soil) on anion-cation balance, organic osmotic regulators, antioxidant enzyme activity, oxidative damage, and nitrogen uptake and utilization in watermelon seedlings under chloride stress of 160 mg(Cl⁻¹)∙kg⁻¹ (oven-dry soil). The test crop was the ‘Jincheng No. 5’ variety of watermelon. The results showed that nitrogen application considerably reduced Cl⁻ and Na⁺ contents in the roots, stems, and leaves of watermelon while significantly increased NO₃⁻ and K⁺ contents at P<0.05; thus the ratios of Cl⁻/NO₃⁻ and Na⁺/K⁺ of the whole plant decreased by 46.0%−69.5% and 31.0%−54.3% compared with that of 0 g∙kg⁻¹ nitrogen rate, respectively. Moreover, the contents of soluble sugar and proline, the activities of superoxide dismutase (SOD) and catalase (CAT) in leaves all reached the maximum levels at 0.15 g∙kg⁻¹ N, which statistically increased by 75.6%, 70.1%, 55.8%, and 54.8% at P<0.05 compared with those at 0 g∙kg⁻¹ N, respectively; while the content of malondialdehyde (MDA) significantly decreased by 59.3%. Moreover, when nitrogen was applied at 0.15 g∙kg⁻¹, the nitrogen accumulation of watermelon increased by 157.7%, activity of nitrate reductase (NR) increased by 62.4%, and nitrogen uptake efficiency and nitrogen use efficiency were 26.25% and 97.10%, respectively. Thus, the fresh and dry weights of the plant increased by 96.9% and 29.0% at P<0.05, respectively. Cluster and correlation analyses of nitrogen application rate and physiological growth indexes of watermelon showed that the mitigation effect of nitrogen application on watermelon chloride stress was 0.15 g∙kg⁻¹ > 0.20 g∙kg⁻¹ > 0.10 g∙kg⁻¹ > 0.25 g∙kg⁻¹. There was significant positive correlation between biomass and dry matter accumulation with nitrogen uptake, utilization efficiency, and nitrogen accumulation; while there were also significant positive correlations between nitrogen accumulation and antioxidant enzyme activity and osmo-modulator content, and negative correlations with Na⁺/K⁺ ratio, Cl⁻/NO₃⁻ ratio, and MDA content. Based on the curve fitting results of each index, the nitrogen application rate of 0.14−0.18 g∙kg⁻¹ was available for the growth and physiological activity of watermelon when the chloride concentration was 160 mg(Cl⁻¹)∙kg⁻¹(oven-dry soil). This indicates that appropriate nitrogen application under chloride salt stress can maintain ion homeostasis in plants by adjusting the Na⁺/K⁺ and Cl⁻/NO₃⁻ ratios, as well as improve the content of osmoregulatory substances and antioxidant enzyme activities, thereby reducing cell membrane oxidative damage, enhancing the physiological resistance of watermelon plants, and achieving a regulatory effect on chloride stress.