Verticillium wilt is an important disease that seriously affects the quality and yield of cotton. Fungal vascular diseases caused by Verticillium dahliae hinders the sustainable development of cotton cultivation. The most effective strategy for managing Verticillium wilt in cotton involves identifying resistance genes, investigating resistance mechanisms, and developing resistant varieties. In the laboratory, in our previous work, V. dahliae strain Vd080 was inoculated into both disease-resistant and disease-susceptible cotton strains, followed by a comprehensive transcriptomic analysis. The findings confirms the correlation between the gene GhCKA1 and disease resistance. In this study, silencing GhCKA1 expression led to reduced levels of reactive oxygen species, callose, and xylem accumulation, thereby inhibiting various defense responses and reducing cotton resistance to V. dahliae. Furthermore, we observed increased resistance to pathogens in Arabidopsis thaliana overexpressing GhCKA1. Subcellular localization experiments in tobacco indicated that GhCKA1 is localized within the nucleus. GUS staining analysis showed that the expression of the GhCKA1 promoter was influenced by pathogenic microorganisms. Additionally, we found that GhCKA1 interacts with aspartic proteases, an important proteolytic enzymes that play significant roles in metabolism and biological regulation. In conclusion, GhCKA1 enhances the resistance of cotton to V. dahliae and interacted with GhAsp1. Therefore, GhCKA1 may be a suitable molecular target to improve the resistance of cotton to Verticillium wilt, and provide a new breeding method for cotton to resist Werticillium wilt.