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Lung cancer remains the leading cause of cancer-related deaths worldwide. Traditional Chinese medicine (TCM) is a valuable resource of active natural products and plays an important role in cancer treatment with the advantages of high efficiency and safety. Wenxia Changfu formula (WCF) is modified from Dahuang Fuzi decoction from Han Dynasty and has been used for treating lung cancer in China. Our previous research showed that WCF had an antitumor effect in vivo and in vitro, while the mechanism has not been well illustrated. In this study, the effect of WCF on the proliferative ability in three lung cancer cells and one noncancerous human cell line was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. WCF suppressed A549, H460, and PC-9 cell viability in a dose-dependent manner, with no inhibition of noncancerous MRC-5 cells after 48 h treatment with WCF (0–50 mg/mL). Furthermore, we screened for genes in A549 cells using four WCF-treated samples and four control samples on a gene expression profile microarray. 21 genes were significantly downregulated by WCF, which may potentially play an important role in the proliferation of A549 cells. High-content screening evaluated whether silencing the 21 genes affected A549 cell growth. The results showed that PIF1 knockdown exhibited the most potent inhibition of cell proliferation compared with the other genes. Downregulation of PIF1 increased A549 cell apoptosis and the activity of caspase 3/7. Besides, RT-PCR showed that the expression levels of PIF1 mRNA decreased significantly in A549, H460, and PC-9 cells after WCF treatment. In conclusion, the present observations indicate that WCF may inhibit lung cancer cell proliferation by promoting apoptosis via regulating the expression of PIF1. [ABSTRACT FROM AUTHOR]