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The calmodulin-binding protein 60 (CBP60) family is a gene family unique to plants, and its members play a crucial role in plant defense responses to pathogens and growth and development. Considering that cotton is the primary source of natural cotton textile fiber, the functional study of its CBP60 gene family members is critical. In this research, we successfully identified 162 CBP60 members from the genomes of 21 species. Of these, 72 members were found in four cotton species, divided into four clades. To understand the function of GhCBP60B in cotton in depth, we conducted a detailed analysis of its sequence, structure, cis-acting elements, and expression patterns. Research results show that GhCBP60B is located in the nucleus and plays a crucial role in cotton growth and development and response to salt and drought stress. After using VIGS (virus-induced gene silencing) technology to conduct gene silencing experiments, we found that the plants silenced by GhCBP60B showed dwarf plants and shortened stem nodes, and the expression of related immune genes also changed. In further abiotic stress treatment experiments, we found that GhCBP60B-silenced plants were more sensitive to drought and salt stress, and their POD (peroxidase) activity was also significantly reduced. These results imply the vital role of GhCBP60B in cotton, especially in regulating plant responses to drought and salt stress. This study systematically analyzed CBP60 gene family members through bioinformatics methods and explored in depth the biological function of GhCBP60B in cotton. These research results lay a solid foundation for the future use of the GhCBP60B gene to improve cotton plant type and its drought and salt resistance. [ABSTRACT FROM AUTHOR]