Phospholipase D (PLD) may play an important role in the infection and inflammation process, but it is unknown whether cellular PLD was activated during the invasion of Listeria monocytogenes into Vero cells. Our study demonstrated that firstly the Vero cells either infected by Listeria monocytogenes or stimulated by PMA shows remarkable increase on PLD activity, compared to basal ones (no stimuli). Further, in the Vero cells with the overexpression of catalytically inactive PLD₂ (mPLD₂-K758R), PLD activition by both Listeria monocytogenes and PMA were dramatically inhibited, leaving basal PLD activity unaffected. These results indicated that the PLD activities may increase while Listeria monocytogenes invade the Vero cells and PLD₂ is likely to be activated. The important physiological role of PLD in this pathogen_host interaction process need to be further studied.