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Abstract The escalating global climate change significantly threatens plant growth, development, and production through salinity stress. Flavonoids, a crucial category of secondary metabolites, have been extensively studied for their role in modulating plant growth and development mechanisms in the face of biological and abiotic stress. The flavonol synthetase ( FLS ) gene plays a key role in the biosynthesis and accumulation of flavonoids. To investigate the correlation between salt tolerance and flavonol synthesis, JsFLS5 was overexpressed in the callus of Juglans sigillata cv. “Qianhe‐7.” This study shows that the upregulation of JsFLS5 significantly elevates the overall flavonoid content by modulating the expression of genes associated with flavonoid synthesis under salinity stress conditions. Additionally, the overexpressing callus exhibited enhanced resistance to salt stress compared to the wild‐type callus, as evidenced by reduced levels of reactive oxygen species accumulation, electrolyte leakage, and malondialdehyde content in the overexpressing callus relative to the wild type (WT). Moreover, the overexpressing callus showed higher antioxidant enzyme activity and a more efficient ascorbic acid‐glutathione cycle. Furthermore, the concentration of Na + in the overexpressing callus was lower than WT, resulting in a decreased Na + /K + ratio. These findings suggest that JsFLS5 overexpression in calli effectively mitigates the oxidative damage induced by osmotic stress and reduces Na + toxicity by enhancing flavonoid synthesis under salt stress conditions. Consequently, this study offers a novel perspective for comprehending the role of JsFLS5 in the response to abiotic stress in J. sigillata .