Being as a significant signal molecule, extracellular ATP (eATP) participates in the processes of plant growth, development and stress responses. These processes are shown to be associated with vesicular trafficking, however, the function of eATP on vesicular trafficking is not yet clear. Here, we explored the effect of eATP on vesicular trafficking in Populus euphratica cells using FM1-43, a selective fluorescent dye for membrane binding. FM1-43 staining revealed that 50 mol/L eATP had no obvious effect on endocytosis in P. euphratica cells. However, the endocytosis was inhibited by higher dosages of eATP (200, 400 mol/L), and the inhibition effect was associated with increasing eATP concentrations. Moreover, high dosages of eATP (200, 400 mol/L) also inhibited exocytosis in P. euphratica cells. In contrast to eATP, ADP (50, 200, 400 mol/L) did not exert a significant impact on vesicular trafficking. This suggests that the inhibitory effect of high concentrations of eATP was not caused by the hydrolyzing product of eATP. Pharmacological experiments showed that the ATP receptor inhibitor, PPADS (100 mol/L), could block the inhibitory effect of high eATP on vesicular trafficking. We conclude that eATP suppresses vesicular trafficking by a purine receptor-mediated signal pathway. However, another purine receptor inhibitor, suramin (100 mol/L), has no obvious inhibitory effect on eATP signaling. Thus it could be inferred that other kinds of P2X cognate receptors mediate inhibitory effect of high eATP on vesicular trafficking. Collectively, eATP can regulate vesicular trafficking in P. euphratica cells, but high concentrations of eATP display a negative regulation on vesicular trafficking.