Cotton is the most economically important natural fiber crop grown in more than sixty-five countries of the world. Fiber length is the main factor affecting fiber quality, but the existing main varieties are short in length and cannot suit the higher demands of the textile industry. It is necessary to discover functional genes that enable fiber length improvement in cotton through molecular breeding.In this study, overexpression of GhEB1C in Arabidopsis thaliana significantly promotes trichomes, tap roots, and root hairs elongation. The molecular regulation of GhEB1C involves its interactions with itself and GhB'ETA, and the function of GhEB1C regulation mainly depends on the two cysteine residues located at the C-terminal. In particular, the function activity of GhEB1C protein triggered with the regulation of protein phosphatase 2A, while silencing of GhEB1C in cotton significantly influenced the fiber protrusions and elongation mechanisms., Further, influenced the expression of MYB-bHLH-WD40 complex, brassinosteroids, and jasmonic acid-related genes, which showed that transcriptional regulation of GhEB1C is indispensable for cotton fiber formation and elongation processes.Our study analyzed the brief molecular mechanism of GhEB1C regulation. Further elucidated that GhEB1C can be a potential target gene to improve cotton fiber length through transgenic breeding.