We explored the role of a chromatin-associated nuclear protein high mobility group protein B1 (HMGB1) in apoptotic response to widely used anticancer drugs. A murine fibroblast model system generated from Hmgb1(+)(/)(+) and Hmgb1(-/-) mice was used to assess the role of HMGB1 protein in cellular response to anticancer nucleoside analogs and precursors, which act without destroying the integrity of DNA. Chemosensitivity experiments with 5-fluorouracil, cytosine arabinoside (araC), and mercaptopurine (MP) demonstrated that Hmgb1(-/-) mouse embryonic fibroblasts (MEFs) were 3 to 10 times more resistant to these drugs compared with Hmgb1(+)(/)(+) MEFs. Hmgb1-deficient cells showed compromised cell cycle arrest and reduced caspase activation after treatment with MP and araC. Phosphorylation of p53 at Ser12 (corresponding to Ser9 in human p53) and Ser18 (corresponding to Ser15 in human p53), as well as phosphorylation of H2AX after drug treatment, was reduced in Hmgb1-deficient cells. trans-Activation experiments demonstrated diminished activation of proapoptotic promoters Bax, Puma, and Noxa in Hmgb1-deficient cells after treatment with MP or araC, consistent with reduced transcriptional activity of p53. We have demonstrated for the first time that Hmgb1 is an essential activator of cellular response to genotoxic stress caused by chemotherapeutic agents (thiopurines, cytarabine, and 5-fluorouracil), which acts at early steps of antimetabolite-induced stress by stimulating phosphorylation of two DNA damage markers, p53 and H2AX. This finding makes HMGB1 a potential target for modulating activity of chemotherapeutic antimetabolites. Identification of proteins sensitive to DNA lesions that occur without the loss of DNA integrity provides new insights into the determinants of drug sensitivity in cancer cells.