Background: Sophora flavescens, a traditional Chinese herb medicine, has been used to prevent and cure type 2 diabetes mellitus (T2DM) both in folk medicine and medical institutions. Modern pharmacological studies have also demonstrated that the flavonoids obtained from Sophora flavescens ethyl acetate extract (SFE) exhibited potential anti-diabetic activity. Our previous study elucidated that SFE exerts anti-T2DM effects by regulating the host–microbial metabolic axis. In the present study, we further explored the pharmacodynamic effect and the potential targets of the anti-T2DM activity of SFE by integrating network pharmacology and pharmacological evaluations. Methods: The diabetic rat model was created by streptozotocin and oral administration with SFE for 8 weeks. Then, the T2DM-related index was estimated to assess the interventional effect of SFE. Network pharmacology was applied to identify the likely targets and the pathways modulated by SFE components. Furthermore, Western blotting was applied to verify the prediction. Results: Pharmacological evaluation in vivo revealed that SFE could markedly improve the blood glucose, serum insulin secretion, insulin resistance, liver glycogen synthesis, and the liver tissue structure in T2DM rats. Through network pharmacology analysis, 101 active compounds of SFE and 114 targets belonging to 128 pathways were identified. The insulin, TNF, IL-6, and PI3K/Akt pathway may be the key targets and pathway. Based on the results of network pharmacology analysis, IRS/PI3K/Akt and IKK/NF-κB/TNF pathways were selected for further validation. Subsequently, experimental results of Western blotting confirmed that SFE may exert anti-T2DM effects by modulating the IRS/PI3K/Akt and IKK/NF-κB/TNF pathways. Conclusion:SFE may protect the T2DM rats by relieving the insulin resistance and inflammation through regulating the IRS/PI3K/Akt and IKK/NF-κB/TNF pathways. The results of the present study would improve the comprehension of the pharmacological basis of SFE against T2DM and provide a theoretical basis for the clinical use of Sophora flavescens.