Zhenjiang aromatic vinegar is a famous traditional fermented cooking ingredient in China, with multiple nutritional and medicinal applications. Zhenjiang aromatic vinegar extract (100–400 μg/mL) is rich in polyphenols increased the glucose uptake and glucose consumption in high glucose-induced insulin resistant HepG2 (IR-HepG2) cells. Zhenjiang aromatic vinegar extract enhanced glycogen synthesis and attenuated gluconeogenesis by regulating key enzymes in IR-HepG2 cells. In addition, Zhenjiang aromatic vinegar extract ameliorated high glucose-induced IR by inhibiting phosphorylated insulin receptor substrate-1 (IRS-1) expression and activating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in IR-HepG2 cells. Moreover, Zhenjiang aromatic vinegar extract reduced reactive oxygen species generation and phosphorylated c-Jun NH2 terminal kinase (JNK) expression in IR-HepG2 cells. The attenuation of the high glucose is owned to the PI3K/Akt pathway activation, glycogen synthesis induction and gluconeogenesis suppression in IR-HepG2 cells.