We compared cold-acclimated and non-acclimated phospholipase D (PLD)δ-knockout and wild-type Arabidopsis thaliana plants for tolerance to freezing. PLDδ-knockout plants showed lower freezing tolerance than did wild-type plants with cold acclimation; without cold acclimation, the two genotypes did not differ in freezing tolerance. Therefore, the PLDδ gene is involved in the process of cold acclimation. The pathways through which PLDδ acts in the process of cold acclimation were then analyzed. During cold acclimation, PLDδ was not involved in the activation of SOD, CAT, or POD anti-oxidative enzymes; it negatively regulated the accumulation of proline and soluble sugar, and mediated the process of freezing resistance induced by the low temperature signaling molecule ABA.