Abstract: This study analyzed the effects of brassinolide (BL) on Na⁺ accumulation, leaf physiological characteristics and differentially expressed genes (DEGs) of cotton leaves under NaCl stress. The results showed that NaCl stress increased the Na⁺, proline and MDA content in the leaves of Sumian 12 and Sumian 22, and changed the expression level of genes in cotton leaves. The application of BL counteracted the NaCl stress-induced growth inhibition in the two tested cotton cultivars. It reduced the accumulation of Na⁺, enhanced proline content, and resulted in a decrease in the MDA content of NaCl-stressed leaves, and the influence of BL on salt-stressed Sumian 12 plants was more ronounced than that on Sumian 22. The digital gene expression analysis in Sumian 12 indicated that BL application significantly influenced the gene expression in NaClstressed cotton leaves, the gene expression pattern as a result of the root applied BL on NaCl-stressed cotton treatment (BL+NaCl) was similar to the normal cotton plants (CK). Our results indicated that brassinolide alleviated NaCl stress on cotton through improving leaf physiological characteristics and gene expression, and resulted in an increase in biomass of NaCl-stressed cotton.