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Summary: Inositol-1,4,5-trisphosphate (IP3) is an important second messenger and one of the products of phosphoinositide-specific phospholipase C (PIPLC)-mediated phosphatidylinositol (4,5) bisphosphate (PIP2) hydrolysis. However, the function of IP3 in cotton is unknown. Here, we characterized the function of GhPIPLC2D in cotton fiber elongation. GhPIPLC2D was preferentially expressed in elongating fibers. Suppression of GhPIPLC2D transcripts resulted in shorter fibers and decreased IP3 accumulation and ethylene biosynthesis. Exogenous application of linolenic acid (C18:3) and phosphatidylinositol (PI), the precursor of IP3, improved IP3 and myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) accumulation, as well as ethylene biosynthesis. Moreover, fiber length in GhPIPLC2D-silenced plant was reduced after exogenous application of IP6 and ethylene. These results indicate that GhPIPLC2D positively regulates fiber elongation and IP3 promotes fiber elongation by enhancing ethylene biosynthesis. Our study broadens our understanding of the function of IP3 in cotton fiber elongation and highlights the possibility of cultivating better cotton varieties by manipulating GhPIPLC2D in the future.