CARF (Collaborator of ARF) was first cloned as an ARF-interacting protein and shown to regulate the p53-p21WAF1-HDM2 pathway, a central to tumor suppression via senescence and apoptosis. Over-expression of CARF induced tumor suppressor functions of p53 resulting in premature senescence of normal human fibroblasts and growth arrest of cancer cells. On the other hand, CARF inhibition in cancer cells led to polyploidy and caspase-dependent apoptosis. In order to determine the mechanism of CARF silencing-induced apoptosis, we examined various cell death and survival pathways including the mitochondrial stress, ATM/ATR, Ras/MAP kinase and retinoblastoma cascades in cultured CARF-compromised cancer cells. We found that CARF is a pleiotropic regulator with widespread effects; its suppression affected all investigated pathways. Most remarkably, CARF-knockdown elicited DNA damage response as evidenced by increased levels of phosphorylated ATM and γ H2AX, leading to induction of mitotic arrest and apoptosis. We demonstrate that CARF is an essential gene that regulates the DNA damage response of cells.