In this report, we studied the role of DNA damage signaling pathway in shiga toxin (STX)-induced mammalian cell death. Shiga toxin 1 exhibited cytotoxic activity in different mammalian cells such as HeLa cells, mouse embryo fibroblasts, and Caco-2 cells (a human intestinal primary fibroblast cell line). STX-1 was found to induce the release of cytochrome c from the mitochondria, nuclear condensation, and fragmentation of chromosomal DNA. STX-1 activated DNA damage signaling as determined by induction of H2AX phosphorylation and cleavage of PARP. Inhibition of caspase-3 reduced STX-1-induced phosphorylation of H2AX and nuclear condensation. It was also found that STX-1-induced p53 expression, and activated ATM in mammalian cells. STX-1-induced nuclear condensation significantly reduced in p53-, and ATM-knockout cells suggesting an involvement of p53 and ATM in transducing signals produced by STX in inducing apoptosis in mammalian cells. This is the first demonstration of involvement of ATM/p53 in STX-inducing mammalian cell death.