Beta-carotene acts as an antioxidant or a pro-oxidant depending on the concentrations that cells are treated with. Oxidative DNA damage is related to apoptosis of various cells. Ataxia-telangiectasia-mutated (ATM), a sensor for DNA-damaging agents, activates a variety of effectors in multiple signaling pathways, such as DNA repair and apoptosis. In the present study, we investigated whether a high concentration of beta-carotene induces apoptosis of gastric adenocarcinoma (AGS) cells and whether ATM is involved in beta-carotene-induced apoptosis of AGS cells. We found that beta-carotene (100 micromol/L) induced apoptosis (determined by cell viability), DNA fragmentation, and the protein levels of p53 and Bcl-2 in AGS cells. ATM levels in the nucleus decreased from beta-carotene in AGS cells. beta-Carotene-induced alterations, including an increase in DNA fragmentation and p53 levels and a decrease in nuclear ATM and cellular Bcl-2 levels, were inhibited in the cells transfected with full-length ATM cDNA compared to wild-type cells or the cells transfected with control vector plasmid control DNA vector (pcDNA). In conclusion, beta-carotene induces apoptosis by increasing apoptotic protein p53 and decreasing anti-apoptotic Bcl-2 as well as nuclear ATM in AGS cells. Nuclear loss of ATM may be the underlying mechanism of beta-carotene-induced apoptosis of gastric cancer cells.