*In Arabidopsis thaliana, the amino acid sequences of membrane-associated acyl-CoA-binding proteins ACBP1 and ACBP2 are highly conserved. We have shown previously that, in developing seeds, ACBP1 accumulates in the cotyledonary cells of embryos and ACBP1 is proposed to be involved in lipid transfer. We show here by immunolocalization, using ACBP2-specific antibodies, that ACBP2 is also expressed in the embryos at various stages of seed development in Arabidopsis. *Phenotypic analyses of acbp1 and acbp2 single mutants revealed that knockout of either ACBP1 or ACBP2 alone did not affect their life cycle as both single mutants exhibited normal growth and development similar to the wild-type. However, the acbp1acbp2 double mutant was embryo lethal and was also defective in callus induction. *On lipid and acyl-CoA analyses, the siliques, but not the leaves, of the acbp1 mutant accumulated galactolipid monogalactosyldiacylglycerol and 18:0-CoA, but the levels of most polyunsaturated species of phospholipid, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine, declined. *As recombinant ACBP1 and ACBP2 bind unsaturated phosphatidylcholine and acyl-CoA esters in vitro, we propose that ACBP1 and ACBP2 are essential in lipid transfer during early embryogenesis in Arabidopsis.