In plant cells, glycerol 3-phosphate (G3P) is apparently able to permeate the plastid envelope, but no specific transporter has been characterized so far. The Arabidopsis five G3Pp proteins have been predicted as putative G3P permeases because of their high homologies with the prokaryotic G3P/phosphate antiporter GlpT. In the present study, G3Pp4 was characterized in detail utilizing reverse genetic approaches. Promoter analysis using GUS expression revealed that G3Pp4 was expressed strongly throughout the embryos during late developmental stages, and the seed lipid contents decreased in two g3pp4 knockout mutants. An enhanced yellow fluorescent protein-fused G3Pp4 was localized in the plastids, functioned physiologically in A. thaliana, and had G3P-transport activity in E. coli. These results suggest that Arabidopsis G3Pp4 is a plastid envelope-localized G3P transporter and involved in accumulation of storage lipids in late embryogenesis.