Background and aim: The ingredients viz., Artemisia roxburghiana, Cissampelos pareira, Stephania glabra, Drimia indica, Roylea cinerea, Tinospora sinensis and Curcuma longa of the present formulation are used to treat diabetes in the Indian traditional medical system. Adopting the concept of multiple herbal mixtures for better therapeutic effects from the ancient Ayurvedic text Sarangdhar Samhita, the present study aimed to develop a polyherbal formulation (PHF) of seven herbs and to evaluate its sodium-glucose cotransporter protein-2 (SGLT2) inhibitory effect on type 2 diabetic rats. Experimental procedure: Streptozotocin (STZ) (60 mg/kg) and nicotinamide (NAM) (120 mg/kg) were intraperitoneally administered to induce type 2 diabetes in Wistar rats. The animals were divided into 5 groups viz. normal control, diabetic control, positive control (dapagliflozin at 0.1 mg/kg) and two test groups (PHF at 250 and 500 mg/kg). Various parameters including blood glucose, serum glutamic pyruvic transaminase (SGPT), serum glutamic-oxaloacetic transaminase (SGOT), bilirubin, triglycerides and creatinine were measured. Results and conclusion: The treatment with PHF (250 and 500 mg/kg) showed a significant (p < 0.05) decrease in blood glucose levels by 56.37% and 58.17%, respectively. The levels of SGOT, SGPT and bilirubin were significantly reduced in PHF-fed diabetic rats. Histopathological examination revealed no major changes in the treated groups as compared to the normal control. The molecular docking study showed strong binding of β-sitosterol, insulanoline, warifteine, dehydrocorydalmine, taraxerol acetate, lupeol, corydalmine and luteolin to SGLT2 protein. The present study concludes that PHF has promising antidiabetic activity via inhibiting SGLT2 protein without showing any adverse effects. © 2022 Center for Food and Biomolecules, National Taiwan University