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Strigolactone (SL) is a plant hormone required for plant development. DWARF53 (D53) functions as a transcription repressor in SL signaling. However, the role of D53 in cotton (Gossypium hirsutum, Gh) fiber development remains unclear. Here, we identify that GhD53 suppresses fiber elongation by repressing transcription of GhFAD3 genes, which control linolenic acid (C18:3) biosynthesis. Mechanistically, GhD53 interacts with SL-related transcriptional activate factor (GhSLRF) to prevent its binding on Omega-3 fatty acid desaturase gene (GhFAD3) promoters, thereby inhibiting GhFAD3 transcription. Upon SL exposure, GhD53 is degraded and leads to GhSLRF activation. This activation further promotes GhFAD3 transcription, C18:3 biosynthesis, and fiber elongation. Our findings identify the molecular mechanism of how SL controls cell elongation via D53 and offer potential strategies to improve cotton quality through SL application.