The health of humans has been negatively impacted by PM exposure, but the chemical composition and toxicity of PM might vary depending on its source. To investigate the toxic effects of particulate matter from different sources on lung epithelial cells (A549), PM samples were collected from residential, industrial, and transportation areas in Nanjing, China. The chemical composition of PM was analyzed, and toxicological experiments were conducted. The A549 cells were exposed using an air-liquid interface (ALI) exposure system, and the cytotoxic indicators of the cells were detected. The research results indicated that acute exposure to different sources of particulate matter at the air-liquid interface caused damage to the cells, induced the production of ROS, caused apoptosis, inflammatory damage, and DNA damage, with a dose-effect relationship. The content of heavy metals and PAHs in PM from the traffic source was relatively high, and the toxic effect of the traffic-source samples on the cells was higher than that of the industrial- and residential-source samples. The cytotoxicity of particulate matter was mostly associated with water-soluble ions, carbon components, heavy metals, PAHs, and endotoxin, based on the analysis of the Pearson correlation. Oxidative stress played an important role in PM-induced biological toxicity.