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Cotton is one of the most important economic crops in the world and is a major source of fiber in the textile industry. Strigolactones (SLs) are a class of carotenoid-derived plant hormones involved in many processes of plant growth and development; however, SL functions in fiber development remain largely unknown. Here, we found that the endogenous SLs were significantly higher in fibers 20 days post-anthesis (DPA). Exogenous SLs significantly increased fiber length and cell wall thickness. Furthermore, we cloned three key SLSbiosynthetic genes, namely GhD27, GhMAX3, and GhMAX4, which were highly expressed in fibers, and subcellular localization analyses revealed that GhD27, GhMAX3, and GhMAX4 were localized in the chloroplast. The exogenous expression of GhD27, GhMAX3, and GhMAX4complemented the physiological phenotypes of d27, max3, and max4mutations in Arabidopsis, respectively. Knockdown of GhD27, GhMAX3, and GhMAX4in cotton resulted in an increased number of axillary buds and leaves, decreased fiber length, and significantly reduced fiber thickness. These findings revealed that SLs participate in plant growth, fiber elongation, and secondary cell wall formation in cotton. These results provide new and effective genetic resources for improving cotton fiber yield and plant architecture.