Changes in membrane lipid composition play important roles in plant adaptation to and survival after freezing. Plant response to cold and freezing involves three distinct phases: cold acclimation, freezing, and post-freezing recovery. Considerable progress has been made toward understanding lipid changes during cold acclimation and freezing, but little is known about lipid alteration during post-freezing recovery. We previously showed that phospholipase D (PLD) is involved in lipid hydrolysis and Arabidopsis thaliana freezing tolerance. This study was undertaken to determine how lipid species change during post-freezing recovery and to determine the effect of two PLDs, PLDalpha1 and PLDdelta, on lipid changes during post-freezing recovery. During post-freezing recovery, hydrolysis of plastidic lipids, monogalactosyldiacylglycerol and plastidic phosphatidylglycerol, is the most prominent change. In contrast, during freezing, hydrolysis of extraplastidic phospholipids, phosphatidylcholine and phosphatidylethanolamine, occurs. Suppression of PLDalpha1 decreased phospholipid hydrolysis and phosphatidic acid production in both the freezing and post-freezing phases, whereas ablation of PLDdelta increased lipid hydrolysis and phosphatidic acid production during post-freezing recovery. Thus, distinctly different changes in lipid hydrolysis occur in freezing and post-freezing recovery. The presence of PLDalpha1 correlates with phospholipid hydrolysis in both freezing and post-freezing phases, whereas the presence of PLDdelta correlates with reduced lipid hydrolysis during post-freezing recovery. These data suggest a negative role for PLDalpha1 and a positive role for PLDdelta in freezing tolerance.