AbstractDiabetic cardiomyopathy (DCM), a diabetes complication, accounts for diabetes‐associated morbidity, mortality, and heart failure. Biflavonoids have been demonstrated to possess extensive pharmacological properties, such as antidiabetic and antioxidant activities. Our study aimed to explore the effects of sciadopitysin, a type of biflavonoid, on DCM and the mechanism involved. An experimental cell model was established in AC16 cardiomyocytes by exposure to high glucose (HG). Cell injury was estimated by detecting cell viability and lactate dehydrogenase (LDH) release. Oxidative stress was determined by measuring malondialdehyde (MDA) level and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), and catalase (CAT). Apoptosis was assessed by flow cytometry analysis, caspase‐3/7 activity assay, and Western blot analysis of cytochrome C (Cyt C) expression. Alternation of the phosphatidylinositol‐3 kinase (PI3K)/protein kinase B (PKB)/glycogen synthase kinase‐3β (GSK‐3β) pathway was detected by Western blot. Results showed that HG exposure reduced viability and increased LDH release in AC16 cells, which was abolished by sciadopitysin treatment. Sciadopitysin inhibited HG‐induced oxidative stress, as evidenced by the reduced MDA content, and the increased activities of SOD, CAT, and GSH‐Px. Sciadopitysin suppressed HG‐induced apoptosis, an increase of caspase‐3/7 activity, and Cyt C expression in AC16 cells. Mechanistically, sciadopitysin activated the PI3K/PKB/GSK‐3β pathway under HG stimulation in AC16 cells. Inhibition of PI3K/PKB/GSK‐3β pathway by LY294002 blocked the effects of sciadopitysin on HG‐induced injury, oxidative stress, and apoptosis in AC16 cells. Summarily, sciadopitysin alleviated HG‐caused oxidative stress and apoptosis in cardiomyocytes by activating the PI3K/PKB/GSK‐3β pathway.