Phospholipids (PLs) are emerging as important factors that initiate signal transduction cascades at the plasma membrane. Their distribution within biological membranes is tightly regulated, e.g. by ATP-binding cassette (ABC) transporters, which preferably translocate PLs from the cytoplasmic to the exoplasmic membrane leaflet and are therefore called PL-floppases. Here, we demonstrate that a plant ABC transporter, Lr34 from wheat (Triticum aestivum), is involved in plasma membrane remodeling characterized by an intracellular accumulation of phosphatidic acid and enhanced outward translocation of phosphatidylserine. In addition, the content of phosphatidylinositol 4,5-bisphosphate in the cytoplasmic leaflet of the plasma membrane was reduced in the presence of the ABC transporter. When heterologously expressed in Saccharomyces cerevisiae, Lr34 promoted oil body formation in a mutant defective in PL-transfer in the secretory pathway. Our results suggest that PL redistribution by Lr34 potentially affects the membrane-bound proteome and contributes to the previously reported stimuli-independent activation of biotic and abiotic stress responses and neutral lipid accumulation in transgenic Lr34-expressing barley plants.