Zilongjin (ZLJ) tablet, which is a traditional Chinese medicine, has been approved as a new anti-tumor drug by the State Food and Drug Administration of China; however, its anti-cancer mechanisms remain elusive. The goal of this study was to investigate the underlying anti-cancer activities of ZLJ tablet in vitro. In this study, four lung cancer cell lines, A549, H446, H460 and H520, were treated with 2.2 mg/mL of ZLJ solution for 24 h at 37 °C under 5% CO2. RNA was isolated and a microarray experiment using the Affymetrix Human Genome U133 plus 2.0 Array was employed to differentiate the expression patterns of cancer-related genes after drug treatment. Of 483 genes in 63 functional categories and 25 different pathways that showed at least a 2-fold change of expression level in the four cancer cell lines, 170 genes were upregulated, and 313 genes were downregulated. Eleven of the 483 genes were cancer-related and belong to the three known pathways: apoptosis, cell cycle regulation and mitogen-activated protein kinase (MAPK) cascade. The microarray data were validated by real-time RT-PCR. The results of this investigation suggest possible anti-cancer mechanisms of the ZLJ tablet, and lay a foundation to further analyse its therapeutic roles. Copyright © 2011 John Wiley & Sons, Ltd.