Metabolites are important for cell fate determination. Fructose‐1,6‐bisphosphate (F1,6P) is the rate‐limiting product in glycolysis and the rate‐limiting substrate in gluconeogenesis. Here, it is discovered that the nuclear‐accumulated F1,6P impairs cancer cell viability by directly binding to high mobility group box 1 (HMGB1), the most abundant non‐histone chromosome structural protein with paradoxical roles in tumor development. F1,6P disrupts the association between the HMGB1 A‐box and C‐tail by targeting K43/K44 residues, inhibits HMGB1 oligomerization, and stabilizes P53 protein by increasing P53–HMGB1 interaction. Moreover, F1,6P lowers the affinity of HMGB1 for DNA and DNA adducts, which sensitizes cancer cells to chemotherapeutic drug(s)‐induced DNA replication stress and DNA damage. Concordantly, F1,6P resensitizes cancer cells with chemotherapy resistance, impairs tumor growth and enhances chemosensitivity in mice, and impedes the growth of human tumor organoids. These findings reveal a novel role for nuclear‐accumulated F1,6P and underscore the potential utility of F1,6P as a drug for cancer therapy.

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