Phospholipase D2 (PLD2), an enzyme involved in vesicle trafficking and membrane signaling, interacts with α-synuclein, a protein known to contribute in the development of Parkinson disease (PD). We previously reported that PLD2 overexpression in rat substantia nigra pars compacta (SNc) causes a rapid neurodegeneration of dopamine neurons, and that α-synuclein suppresses PLD2-induced nigral degeneration (Gorbatyuk et al., 2010). Here, we report that PLD2 toxicity is due to its lipase activity. Overexpression of a catalytically inactive mutant (K758R) of PLD2 prevents the loss of dopaminergic neurons in the SNc and does not show signs of toxicity after 10 weeks of overexpression. Further, mutant K758R does not affect dopamine levels in the striatum. In contrast, mutants that prevent PLD2 interaction with dynamin or growth factor receptor bound protein 2 (Grb2) but retained lipase activity, continued to show rapid neurodegeneration. These findings suggest that neither the interaction of PLD2 with dynamin, which has a role in vesicle trafficking, nor the PLD2 interaction with Grb2, which has multiple roles in cell cycle control, chemotaxis and activation of tyrosine kinase complexes, are the primary cause of neurodegeneration. Instead, the synthesis of phosphatidic acid (the product of PLD2), which is a second messenger in multiple cellular pathways, appears to be the key to PLD2 induced neurodegeneration. The fact that α-synuclein is a regulator of PLD2 activity suggests that regulation of PLD2 activity could be important in the progression of PD.