Arabidopsis thaliana At2g33470 encodes a glycolipid transfer protein (GLTP) that enhances the intervesicular trafficking of glycosphingolipids in vitro. GLTPs have previously been identified in animals and fungi but not in plants. Thus, At2g33470 is the first identified plant GLTP and we have designated it AtGTLP1. AtGLTP1 transferred BODIPY-glucosylceramide at a rate of 0.7 pmol x s(-1), but BODIPY-galactosylceramide and BODIPY-lactosylceramide were transferred slowly, with rates below 0.1 pmol x s(-1). AtGLTP1 did not transfer BODIPY-sphingomyelin, monogalactosyldiacylglycerol or digalactosyldiacylglycerol. The human GLTP transfers BODIPY-glucosylceramide, BODIPY-galactosylceramide and BODIPY-lactosylceramide with rates greater than 0.8 pmol.s(-1). Structural models showed that the residues that are most critical for glycosphingolipid binding in human GLTP are conserved in AtGLTP1, but some of the sugar-binding residues are unique, and this provides an explanation for the distinctly different transfer preferences of AtGLTP1 and human GLTP. The AtGLTP1 variant Arg59Lys/Asn95Leu showed low BODIPY-glucosylceramide transfer activity, indicating that Arg59 and/or Asn95 are important for the specific binding of glucosylceramide to AtGLTP1. We also show that, in A. thaliana, AtGLTP1 together with At1g21360 and At3g21260 constitute a small gene family orthologous to the mammalian GLTPs. However, At1g21360 and At3g21260 did not transfer any of the tested lipids in vitro.