The Glucosyltransferases, Rab-like GTPase activators and Myotubularins (GRAM) domain is highly conserved in eukaryotic cells and expected to be involved in membrane-associated processes. However, the functional characterization of GRAM domain genes in cotton has not yet been achieved. Here, we identified 164 genes encoding GRAM domain proteins in four cotton species, comprising two subfamilies. In Gossypium hirsutum, the transcriptome data showed that GhGRAM31 was predominantly expressed at the rapid elongation stage of fiber development, and may regulate cotton fiber length. GhGRAM31 RNAi transgenic cotton lines showed inhibition of fiber elongation, producing shorter mature fibers, coupled with expression changes of genes related to fiber development. Moreover, lint percentage and seed size were also decreased in RNAi lines. Further analysis revealed that GhGRAM31 directly interacts with another two GRAM domain proteins, GhGRAM5 and GhGRAM35. GhGRAM5 also interacts with the transcription factor GhTTG1, while GhGRAM35 interacts with transcription factors GhHOX1 and GhHD1. Moreover, GhGRAM31 and GhGRAM35 co-expression can promote GhHD1 transcription activity in cotton protoplast. This study provides new insights into the biological function of the GRAM domain gene family in cotton, and we suggest that some GRAM domain genes might be applied to the genetic improvement of fibers.