Accumulating clinical evidence indicates that diabetic liver cancer patients are less sensitive to intra-arterial chemotherapy than non-diabetic cancer patients. However, the underlying mechanism remains largely uncharacterized. Here, we report that hyperglycemia inhibits AMPK pathway and subsequently reduces adriamycin (ADR)-induced DNA damage, resulting in decreased chemotherapeutic sensitivity of ADR. HepG2 and Bel-7402 cells were treated with ADR in various glucose conditions and then subjected to cell proliferation assay and apoptosis. The IC50 of ADR greatly increased with the increasing concentration of glucose (15 ± 4 nM to 93 ± 39 nM in HepG2, 78 ± 8 nM to 1310 ± 155 nM in Bel-7402). Both FACs and Western blot analysis indicated that high concentration of glucose protected cells from ADR-induced apoptosis. Mouse hepatoma H22 xenografts were established both in db/db diabetic mice and STZ-induced diabetic mice. The inhibitory effect in tumor growth of ADR was significantly reduced in diabetic mice, which could be recovered by insulin therapy. Hyperglycemia greatly ameliorated AMPK activation and H2AX expression caused by ADR treatment. Pretreatment with compound C or AMPK silencing eliminated hyperglycemia reduced cytotoxicity of ADR. However, the impaired cytotoxicity in hyperglycemia was recovered by treatment with AMPK activator AICAR. This study indicates that hyperglycemia impairs the chemotherapeutic sensitivity of ADR by downregulating AMPK pathway and reducing ADR-induced DNA damage.