Diabetes mellitus is a metabolic disorder having serious consequences on health and is becoming the 3rd most fatal disease worldwide. The current study was designed to prepare, characterize, standardize and explore the antidiabetic potential of a polyherbal formulation comprising aqueous extracts of Momordica charantia,Syzygium cumini, Acacia nilotica, Elettaria cardamomum, Cicer arietinum L, Foeniculum Vulgare and Gymnema sylvestre. Hyperglycemia was induced by alloxan monohydrate. After induction of diabetes polyherbal formulation was administered in graded doses 200 mg/kg, 400 mg/kg & 600 mg/kg to treated groups I, II and III respectively.Prior to start of In-vivo trial, polyherbal formulation was standardized by performing phytochemical, mineral and proximate analysis. Polyherbal formulation was found to be rich in phytoconstituents and minerals with antihyperglycemic and antioxidant potential. Proximate/physicochemical analysis further confirmed the nutritional value of formulation due to the presence of crude fat, crude fiber, crude protein and carbohydrate contents in formulation. Antihyperglycemic potential of polyherbal formulation was determined through biochemical, histopathological and oxidative stress analysis. Results of the study have revealed that polyherbal formulation significantly reversed the alloxan induced hyperglycemia in rat models by improving the biochemical and oxidative stress parameters in dose dependent manner. Highest dose of polyherbal formulation (600 mg/kg) significantly reduced the serum overall mean glucose (239+21.35), glycosylated hemoglobin (8.16+0.47) and increased the serum overall mean insulin (12.84+1.28) levels in comparison to positive control group having serum overall mean glucose (356.27±6.57), glycosylated hemoglobin (12.34±0.48) and insulin (6.84±0.71) levels respectively. Moreover, histopathological analysis also supported the antidiabetic potential of polyherbal formulation. Polyherbal formulation enhanced the performance of pancreatic β cells by upregulating the expression of Pdx-1, Ins-1 and Ins-2 (insulin signaling cascade) and down regulating the expression of cellular stress cascade, MAPK downstream c-Jun N terminal kinase (JNK). Conclusively, the results of current study indicated the potent hypoglycemic properties of polyherbal formulation.