The precise identities of the virulence factors of Pseudomonas aeruginosa after bacteriolysis are still unknown. In the present study, we identified PA0423 protein, which was isolated from the Pseudomonas PAO-1 strain, as the factor responsible for cytotoxicity in lung epithelial cells. Whole bacterial cell lysate of P. aeruginosa PAO-1 caused cytotoxicity in A549 lung epithelial cells. This cytotoxic factor could be partially purified via gel-filtration and anion-exchange column chromatography, and its activity was attenuated by proteinase K treatment. The cytotoxic fraction increased caspase-3/7 activity in A549 cells, suggesting the induction of apoptosis. This fraction was then subjected to amino-acid sequence analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, resulting in the identification of 7 matches, 4 of which were with known proteins (PA0122, PA2687, PA3406, and PA0423). Deletion mutant analysis of these 7 candidates revealed that only the PA0423 mutation led to reduced cytotoxicity, indicating that this protein is the virulence factor. Furthermore, PA0423 recombinant protein was constructed, purified, and refolded. Transduction of recombinant PA0423, but not PA0122, into A549 cells engendered a dose-dependent cytotoxic effect. These results show the first evidence that specific bacteriolysis-induced virulence factor PA0423 from Pseudomonas is toxic to lung epithelial cells.