Brassinolide (BL) is a plant growth regulator. This study analyzes the effects of BL on cotton growth, Na+ accumulation, proline content, MDA content, antioxidant enzyme activities, and differentially expressed genes (DEGs) of cotton roots under NaCl stress. We grew two cotton cultivars, Sumian 12 (salt sensitive) and Sumian 22 (salt tolerant), in continuously aerated Hoagland’s nutrient solution supplemented with 200 mM NaCl. NaCl stress increased the Na+, proline, and MDA content and decreased root activity and protein content in the roots of Sumian 12 and 22, and the change rang of Sumian 12 was higher than that of Sumian 22. 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 an overall change in the activities of antioxidant enzymes causing a decrease in the MDA content of NaCl-stressed roots, and the influence of BL on salt-stressed Sumian 12 plants was more pronounced 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 roots. The majority of 7659 (3661 up-regulated/3998 down-regulated) DEGs of NaCl/CK in roots of Sumian 12 were regulated by BL, and the gene expression pattern as a result of the root-applied BL on NaCl-stressed cotton treatment (BL + NaCl) was similar to the control. Our results indicate that the rootapplied brassinolide alleviates NaCl stress on cotton through improving root activity, physiology, and gene expression.