Objective: To identify the active ingredients, potential targets, and mechanism of Rhizoma coptidis by bioinformatics method, and to explore the hypoglycemic effect of Rhizoma coptidis by in vitro experiments. Methods: The chemical components of Rhizoma coptidis were collected through database search, and oral bioavailability and drug-likeness were used for preliminary screening. The targets of Rhizoma coptidis and diabetes-related targets were collected by database retrieval and reverse docking techniques, and the biological process of cross-set proteins was analyzed. The inhibitory effects of Rhizoma coptidis on α-glucosidase, α-amylase activity, and advanced glycation end products (AGEs) were determined via in vitro experiments. In addition, the effects of Rhizoma coptidis on pre-adipocyte differentiation, absorption of glucose by adipocytes, and the level of intracellular triglyceride were investigated using the adipocyte differentiation model. Results: There were 11 potentially active ingredients in Rhizoma coptidis. IL-6, caspase-3, epidermal growth factor receptor (EGFR), MYC, and estrogen receptor 1 were considered as the key genes. The bioinformatics analysis showed that Rhizoma coptidis played an anti-diabetic role mainly via biological processes and signaling pathways including hormone receptor activity, glutathione binding, steroid binding, etc. In vitro experiments showed that the extract of Rhizoma coptidis inhibited the activities of α-glucosidase and α-amylase, and the generation of AGEs; meanwhile, the extract promoted the absorption of glucose by adipocytes. In addition, the extract of Rhizoma coptidis decreased triglyceride level. Conclusions: Our network pharmacology and in vitro experiments demonstrate the anti-diabetic effects and possible underlying mechanisms of Rhizoma coptidis extract.