Objective: To further clarify the therapeutic effect of gentiopicrin on AMPK–/– DPN rat models as well as its modification in the expressions of PPAR-γ/AMPK/ACC signaling pathway. Further to evaluate the role of AMPK in DPN pathogenesis and drug intervention. Methods: DPN rat models were induced by streptozotocin (STZ) using AMPK–/– rats. All the rats were randomly divided into 5 groups with 10 rats each: 1) control group; 2) DPN group; 3) AMPK–/– DPN group (STZ-induced DPN model on AMPK–/– rats); 4) gentiopicrin group (DPN+ Gent 20 mg/kg); 5) AMPK–/– intervention group (DPN + Gent 20 mg/kg + AMPK–/–). Weight and blood sugar at the beginning (D0) and the end of experiment (D14) were recorded and measured. Motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) were detected while gene expressions of PPAR-γ/AMPK/ACC signaling pathway were evaluated. Results: After intervention with gentiopicrin, the average body weight increased and blood glucose decreased, the effect of gentiopicrin was weaken in AMPK–/– rat models. There was a significant decrease of MNCV and SNCV in DPN group with abnormal expression of PPAR-γ/AMPK/ACC signaling pathway. Intervened by gentiopicrin, MNCV and SNCV increased, PPAR-γ and AMPK expressions were enhanced, and ACC expression was inhibited (P<0.05). Nevertheless, compared with the general DPN rats with Gent intervention, gentiopicrin administrated in AMPK–/– DPN rats failed to improve the MNCV and SNCV, neither to promote AMPK expression nor inhibit the ACC expression (P<0.05). Conclusion: PPAR-γ affected ACC expression by means of downstream key factor AMPK in PPAR-γ/AMPK/ACC signaling pathway. AMPK acted as an extremely important factor for gentiopicrin exerting therapeutic effect in DPN.