The omega-3 fatty acid desaturase enzyme gene FAD3 is responsible for converting linoleic acid to linolenic acid in plant fatty acid synthesis. Despite limited knowledge of its role in cotton growth, our study focused on GhFAD3-4, a gene within the FAD3 family, which was found to promote fiber elongation and cell wall thickness in cotton. GhFAD3-4 was predominantly expressed in elongating fibers, and its suppression led to shorter fibers with reduced cell wall thickness and phosphoinositide (PI) and inositol triphosphate (IP₃) levels. Transcriptome analysis of GhFAD3-4 knock-out mutants revealed significant impacts on genes involved in the phosphoinositol signaling pathway. Experimental evidence demonstrated that GhFAD3-4 positively regulated the expression of the GhBoGH3B and GhPIS genes, influencing cotton fiber development through the inositol signaling pathway. The application of PI and IP₆ externally increased fiber length in GhFAD3-4 knock-out plants, while inhibiting PI led to a reduced fiber length in GhFAD3-4 overexpressing plants. These findings suggest that GhFAD3-4 plays a crucial role in enhancing fiber development by promoting PI and IP₃ biosynthesis, offering the potential for breeding cotton varieties with superior fiber quality.