Vesicle shuttling is critical for many cellular and organismal processes, including embryonic development. GDI proteins contribute to vesicle shuttling by regulating the activity of Rab GTPases, controlling their cycling between the inactive cytosol and active membrane bound states. While identifying genes controlled by A-form DNA sequences we discovered a previously unknown member of the GDI family, GDI3. The GDI3 gene is found only in amphibians and fish and is developmentally expressed in Xenopus from neurula stages onwards in the neural plate, and subsequently in both dorsal and anterior structures. Depletion or over-expression of the GDI3 protein in Xenopus embryos gives rise to very similar phenotypes, suggesting that strict control of GDI3 protein levels is required for correct embryonic development. Our analysis suggests the evolutionary origins of GDI3 and that it is functionally distinct from GDI1. Predicted structural analysis of GDI3 suggests that the key difference between GDI1 and GDI3 lies in their lipid binding pockets.