Abnormalities in the secretion of insulin are the cause of pathology and complications in diabetic patients. The aim of this study was to investigate the anti-diabetic effect of polysaccharide extracts from the split gill mushroom in type 2 diabetes rats administered a low dose of streptozotocin (STZ) in combination with a high-fat diet. The rats were divided into 6 groups: the control group (ND), the control group fed with polysaccharide extract from split gill (ND240), the diabetes group (HFD+DM), the diabetic group fed 120 (HFD+S120) and 240 mg/kg BW polysaccharide extract (HFD+S240), and the diabetic group receiving metformin (HFD+Met). Subsequently, the Islets of Langerhans of pancreatic tissue were studied using a light microscope and transmission electron microscopy (TEM). Immunofluorescence for the detection of insulin and glucose transporter 2 (GLUT2) proteins, and malondialdehyde (MDA) were also detected in pancreatic tissue. In the diabetic and HFD+120 groups, the tissues harbored various pathologies. The HFD+S240 and HFD+Met groups were found to have lower blood sugar levels. The levels of insulin and GLUT2 increased compared with the diabetic group. Additionally, the levels of MDA were reduced. The use of polysaccharide extract from split gill mushrooms (240 mg/kg BW) is an alternative to treating various pathologies in the relief or treatment of diabetes mellitus.