Abstract The glucosyltransferases, Rab-like GTPase activators and myotubularins (GRAM) domain is highly conserved in eukaryotic cells and is found in proteins involved in membrane-associated processes. GRAM domain proteins have not yet been functionally characterized in cotton. In this study, we identified 164 genes encoding GRAM domain proteins in four cotton species, comprising two subfamilies. In Gossypium hirsutum, our transcriptome data showed that GhGRAM31 was predominantly expressed during the rapid elongation stage of fiber development and that it might control fiber length. GhGRAM31-RNAi transgenic cotton lines showed inhibition of fiber elongation and produced shorter mature fibers, and this was coupled with expression changes of genes related to fiber development. In addition, lint percentage and seed size were also decreased in the RNAi lines. Further examination revealed that GhGRAM31 directly interacts with two other GRAM-domain proteins, GhGRAM5 and GhGRAM35. GhGRAM5 also interacts with the transcription factor GhTTG1, while GhGRAM35 interacts with the transcription factors GhHOX1 and GhHD1. Co-expression of GhGRAM31 and GhGRAM35 was able to promote GhHD1 transcription activity in cotton protoplasts. Our results provide new insights into the biological function of the GRAM-domain protein family in cotton, and selected genes have the potential to be utilized in future programs for the genetic improvement of fibers.