In this study, A549 cells were exposed to PM_2.5 dustfall of different concentrations, respectively. MTT assay was used to evaluate cell viability. Cell morphology was observed through microscope after W-G staining. Fluorescence probe DCFH-DA and JC-1 were applied to label A549 cells after being treated by PM_2.5 to detect intracellular level of ROS and change of MMP, with the purpose of investigating the mitochondrial oxidative damage in A549 cells induced by PM_2.5 dust-fall. The results showed that, compared with the control group, A549 cell viability was (81.77±6.15)% after being exposed to PM_2.5 at 12.5 μg/mL for 3 h, and was declined in a dose-dependent manner and time-dependent effect. It was observed that cell morphology was changed, cell membrane was dissolved and cell micro-nucleus appeared after being treated with PM_2.5. In addition, the ROS levels were significantly higher than those of the control group; with the increase of PM_2.5 concentration, the fluorescence intensity showed an increasing tendency gradually. Simultaneously, the MMP levels were declined in a dose-dependent manner after being treated with PM_2.5. And there was a negative correlation between the ROS level and MMP (R²=0.878). It is inferred that PM_2.5 may stimulate the production of ROS and induce the declining of MMP, resulting in A549 cell mitochondrial oxidative damage.