Phosphorus is an essential macronutrient that often limits plant growth and development. Under phosphorus-limited conditions, plants undergo substantial alterations in membrane lipid composition to cope with phosphorus deficiency. To characterize the changes in lipid species and to identify enzymes involved in plant response to phosphorus starvation, 140 molecular species of polar glycerolipids were quantitatively profiled in rosettes and roots of wild-type Arabidopsis (Arabidopsis thaliana) and phospholipase D knockout mutants pldζ1, pldζ2, and pldζ1pldζ2. In response to phosphorus starvation, the concentration of phospholipids was decreased and that of galactolipids was increased. Phospholipid lost in phosphorus-starved Arabidopsis rosettes was replaced by an equal amount of galactolipid. The concentration of phospholipid lost in roots was much greater than in rosettes. Disruption of both PLDζ1 and PLDζ2 function resulted in a smaller decrease in phosphatidylcholine and a smaller increase in digalactosyldiacylglycerol in phosphorus-starved roots. The results suggest that hydrolysis of phosphatidylcholine by PLDζs during phosphorus starvation contributes to the supply of inorganic phosphorus for cell metabolism and diacylglycerol moieties for galactolipid synthesis.