Objective To investigate the toxic affects of 25 nm TiO2 nanoparticles in anatase crystal against human lung cancer cell line A549 after different exposure periods. Methods A549 cells were incubated with 100 μg/ml TiO2 nanoparticle suspension for 1 h, 4 h, 8 h and 24 h at 37℃ and 5%CO2. Then the damage degrees of cell membrane caused by TiO2 nanoparticle were assessed by LDH assay. ATP assay was used to evaluate the influence of TiO2 nanoparticle on mitochondria. SOD activity test was used to detect the oxidative damage of the cells caused by TiO2 nanoparticles. The cellular ultrastructure changes of A549 cells were observed by transmission electron microscopy. CCK-8 assay was performed to evaluate the viability of TiO2-treated cells after 1 h, 4 h, 8 h and 24 h exposure. Results With the prolongation of exposure time, the activity of extracellular LDH was increased and that of SOD was decreased; the concentration of intracellular ATP was reduced and the survival rate of cells was significantly decreased(P<0.05). The mitochondria and endoplasmic reticulum were swollen and expanded to different degrees. Conclusion TiO2 nanoparticles can result in oxidative damage and inhibit the growth of A549 cells, and the effects are in a time-dependent manner.