Most types of plant phospholipase D (PLD) require Ca2+ for activity, but how Ca2+ affects PLD activity is not well understood. We reported previously that Ca2+ binds to the regulatory C2 domain that occurs in the N terminus of the Ca2+-requiring PLDs. Using Arabidopsis thaliana PLDβ and C2-deleted PLDβ (PLDβcat), we now show that Ca2+ also interacts with the catalytic regions of PLD. PLDβcat exhibited Ca2+-dependent activity, was much less active, and required a higher level of Ca2+ than the full-length PLDβ. Ca2+ binding of the proteins was stimulated by phospholipids; phosphatidylserine was the most effective among those tested. Scatchard plot analysis of Ca2+ binding data yielded an estimate of 3.6 high affinity (Kd = 29 μM) binding sites on PLDβ. The Ca2+-PLDβcat interaction increased the affinity of the protein for the activator, phosphatidylinositol 4,5-bisphosphate, but not for the substrate, phosphatidylcholine. This is in contrast to the effect of Ca2+ binding to the C2 domain, which stimulates phosphatidylcholine binding but inhibits phosphatidylinositol 4,5-bisphosphate binding of the domain. These results demonstrate the contrasting and complementary effects of the Ca2+- and lipid-binding properties of the C2 and catalytic domains of plant PLD and provide insight into the mechanism by which Ca2+ regulates PLD activity.