Agrimonia pilosa Ledeb (AP) has already been applied in practice for the treatment of different disorders and is available to access without the provision of a medical prescription. The present study aims at investigating the effect of bioactive compounds isolated from AP on the improvement of insulin resistance, figuring out the mechanism in insulin-responsive cell lines. Five compounds were isolated from AP using column chromatography, including agrimonolide (K1), desmethylagrimonolide (K2), tormentic acid (K3), ursolic acid (K4), and quercetin (K5). Glucose metabolism was evaluated in insulin-resistant HepG2 cells. Ursolic acid had the strongest activity among all isolated compounds with the lowering value of 71.5% (1.24 mM glucose in DMEM) and 71.7% (1.23 mM) when compared to the control. K1 consisting of K2 effectively increased the insulin-mediated glycogen level in hepatocytes. At a concentration level of 20 μM, K2 significantly elevated the hepatic glucokinase (GK) activity (3.2 U min-1 mg-1 protein), followed by K1 (3.0 U min-1 mg-1 protein). Both of them significantly increased (p < 0.05) the GK activity as compared to the control. On the same lines, K2 and K1 caused a significant reduction of the glucose-6-phosphatase (G6Pase) activity and a significant change in the phosphoenolpyruvate carboxykinase (PEPCK) activity. In summary, bioactive compounds in AP may play an important role in regulating the glucose metabolism in insulin-resistant HepG2 cells and could be developed as a promising natural material for diabetes prevention and treatment.