Background:Excessive hepatic glucose production (HGP) largely promotes the development of type 2 diabetes mellitus (T2DM), and the inhibition of HGP significantly ameliorates T2DM. Huanglian-Renshen-Decoction (HRD), a classic traditional Chinese herb medicine, is widely used for the treatment of diabetes in clinic for centuries and proved effective. However, the relevant mechanisms of HRD are not fully understood. Purpose: Based on that, this study was designed to identify the potential effects and underlying mechanisms of HRD on HGP by a comprehensive investigation that integrated in vivo functional experiments, network pharmacology, molecular docking, transcriptomics and molecular biology. Methods: After confirming the therapeutic effects of HRD on T2DM mice, the inhibitory role of HRD on HGP was evaluated by pyruvate and glucagon tolerance tests, liver positron emission tomography (PET) imaging and the detection of gluconeogenic key enzymes. Then, network pharmacology and transcriptomics approaches were used to clarify the underlying mechanisms. Molecular biology, computational docking analysis and in vitro experiments were applied for final mechanism verification. Results: Here, our results showed that HRD can decrease weight gain and blood glucose, increase fasting insulin, glucose clearance and insulin sensitivity in T2DM mice. Dysregulated lipid profile was also corrected by HRD administration. Pyruvate, glucagon tolerance tests and liver PET imaging all indicated that HRD inhibited the abnormal HGP of T2DM, and the expressions of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) were significantly suppressed by HRD as expected. Network pharmacology and transcriptomics approaches illustrated that PI3K/Akt/FoxO1 signaling pathway may be responsible for the inhibitory effect of HRD on HGP. Afterward, further western blot and immunoprecipitation found that HRD did activate PI3K/Akt/FoxO1 signaling pathway in T2DM mice, which confirmed previous results. Additionally, the conclusion was further supported by molecular docking and in vitro experiments, in which identified HRD compound, oxyberberine, was proven to exert an obvious effect on Akt. Conclusion: Our data demonstrated that HRD can treat T2DM by inhibiting hepatic glucose production, the underlying mechanisms were associated with the activation of PI3K/Akt/FoxO1 signaling pathway.