To study the acute toxic effects of PCBs on airway exposure, the cell viability, apoptosis and mitochondrial functions of human lung cancer cell line A549 were measured and compared after Aroclor 1254 exposure for different time. The results showed that Aroclor 1254 could inhibit cell viability and increase cell apoptosis in a concentration- and time-dependent manner. The mitochondrial apoptosis pathway was confirmed playing an important role. ROS elevation was an early response within 1h treatment of Aroclor 1254. Then after 4?h of Aroclor 1254 exposure, the intracellular calcium level increased and mitochondrial transmembrane potential (ΔΨm) collapsed, accompanying with Cytochrome c (Cyt-c) leakage, boosting expression of Bax, Apaf-1 and miRNA155, which were involved in the mitochondrial apoptosis pathway. After 24?h of Aroclor 1254 exposure, ROS returned to normal level, but cell apoptosis rate was higher than that at 4?h with ΔΨm continued collapsing and intracellular calcium increased. In conclusion, Aroclor 1254 could suppress cell viability and induce apoptosis in A549 cells, which was associated with ROS over-production and elevated cellular Ca(2+) level, which may result in mitochondrial dysfunction, inducing expression of Bax/Cyt-c/Apaf-1 and miRNA155.