Bufalin is a key component of a Chinese medicine (Chan Su) and has been proved effective in killing various cancer cells. Its role in inducing DNA damage and the inhibition of the DNA damage response (DDR) has been reported, but none have studied such action in lung cancer in detail. In this study, we demonstrated bufalin-induced DNA damage and condensation in NCI-H460 cells through a comet assay and DAPI staining, respectively. Western blotting indicated that bufalin suppressed the protein levels associated with DNA damage and repair, such as a DNA dependent serine/threonine protein kinase (DNA-PK), DNA repair proteins breast cancer 1, early onset (BRCA1), 14-3-3 σ (an important checkpoint keeper of DDR), mediator of DNA damage checkpoint 1 (MDC1), O6-methylguanine-DNA methyltransferase (MGMT) and p53 (tumor suppressor protein). Bufalin could activate phosphorylated p53 in NCI-H460 cells. DNA damage in NCI-H460 cells after treatment with bufalin up-regulated its ATM and ATR genes, which encode proteins functioning as sensors in DDR, and also up-regulated the gene expression (mRNA) of BRCA1 and DNA-PK. But bufalin suppressed the gene expression (mRNA) of p53 and 14-3-3 σ, however, bufalin did not significantly affect the mRNA of MGMT. In conclusion, bufalin induced DNA damage in NCI-H460 cells and also inhibited its DNA repair and checkpoint function.