The present study aimed to evaluate the impact of dendrobium mixture (DMix) on the gene and protein expression of insulin signaling pathway-associated factors in the livers of diabetic rats. The molecular mechanisms by which DMix inhibits gluconeogenesis were also investigated. A total of 47 female Wistar rats were used in the present study. Of these, 11 rats were randomly selected as healthy controls and diabetes was induced in the remaining 36 rats by administering a high-fat and high-sugar diet for 6 weeks, followed by two intraperitoneal injections of streptozotocin. The 36 rats were screened for diabetes and then randomly divided into three groups: Model, metformin and DMix groups. Following 12 weeks of treatment, the fasting blood glucose (FBG), glycosylated serum protein (GSP), serum insulin, blood lipids [total cholesterol (Tch) and triglycerides (TG)], alanine transaminase (ALT) and aspartate transaminase (AST) were assessed. In addition, hematoxylin and eosin staining was used for histomorphological examination of the liver tissues. The mRNA expression of insulin receptor (InsR), forkhead box protein O1 (FoxO1), phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) in the liver was measured with reverse transcription-quantitative polymerase chain reaction and the protein expression of InsR, phosphoinositide-3-kinase (PI3K), phosphorylated (p)-PI3K, protein kinase B (Akt), p-Akt, FoxO1, PEPCK and G6Pase in the liver was measured by western blot analysis. The FBG, GSP, InsR, Tch, TG, ALT and AST levels were significantly lower in the DMix-treated group compared with the model group (P<0.05). In addition, DMix treatment notably improved liver histopathology and significantly increased the gene and protein expression of InsR, PI3K and Akt (P<0.05). DMix treatment also significantly reduced the gene and protein expression of FoxO1, PEPCK and G6Pase (P<0.05). DMix effectively reduced FBG and blood lipids and significantly improved liver function and insulin resistance in diabetic rats, possibly by regulating the gene and protein expression of molecules associated with the PI3K/Akt signaling pathway.