This study is the first to investigate the anticancer effect of isoliquiritigenin (ISL) in human cervical carcinoma HeLa cells. The results reveal that ISL inhibits HeLa cells by blocking cell cycle progression in the G2/M phase and inducing apoptosis. Blockade of cell cycle is associated with increased activation of ataxia telangiectasia-mutated (ATM). Activation of ATM by ISL phosphorylated p53 at Serine15, resulting in increased stability of p53 by decreasing p53 and murine double minute-2 (MDM2) interaction. In addition, ISL-mediated G2/M phase arrest was also associated with decreases in the amounts of cyclin B, cyclin A, cdc2, and cdc25C, and increases in the phosphorylation of Chk2, cdc25C, and cdc2. The specific ATM inhibitor caffeine significantly decreased ISL-mediated G2/M arrest by inhibiting the phosphorylation of p53 (Serine15) and Chk2. ISL induced apoptotic cell death is associated with changes in the expression of Bax and Bak, decreasing levels of Bcl-2 and Bcl-X(L), and subsequently triggering mitochondrial apoptotic pathway. In addition, pretreatment of cells with caspase-9 inhibitor blocked ISL-induced apoptosis, indicating that caspase-9 activation is involved in ISL-mediated HeLa cell apoptosis. These findings suggest that ISL may be a promising chemopreventive agent against human uterine cervical cancer.