Phytohormones are thought to play important regulatory roles in the development of cotton fibers. Previous pharmacological experiments indicated that brassinosteroid (BR), along with auxin and gibberellins, promoted cotton fiber elongation. To further evaluate the role of the BR signaling pathway in cotton fiber development, we created transgenic cotton plants that express transgenes designed to over-express or suppress the expression of the BR receptor BRI1. Analysis of the characteristics of cotton fiber from these plants indicates that alteration of BR responsiveness has little effect on fiber length. However, suppression of BRI1 expression strongly inhibits secondary cell wall development, resulting in fibers with reduced maturity. Conversely, over-expression of BRI1 leads to increases in fiber cellulose deposition. These alterations in fiber cell wall development correspond with changes in cellulose synthase gene expression, indicating that secondary wall deposition is regulated, at least in part, by BR-regulated modulation of cellulose synthase gene expression. These results indicate that, while BR signaling does not limit cotton fiber elongation, it is required for normal maturation of cotton fibers through the deposition of cellulose into the secondary cell wall.