The present study was designed to explore the mechanism by which ethanol extract of Bombax ceiba leaves (BCE) and its main constituent mangiferin (MGF) affect diabetic nephropathy by combating oxidative stress. Oral administration of BCE and MGF to normal and streptozotocin (STZ)-induced diabetic mice were carried out. Fasting blood glucose, 24-h urinary albumin, serum creatinine, and blood urea nitrogen were tested, histopathology, and immunohistochemical analysis of kidney tissues were performed. Moreover, mesangial cells were treated with BCE and MGF for 48 h with or without 25 mmol·L −1 of glucose. Immunofluorescence, Western blot and apoptosis analyses were used to investigate their regulation of oxidative stress and mitochondrial function. BCE and MGF ameliorated biochemical parameters and restored STZ-induced renal injury in the model mice. In vitro study showed that high glucose stimulation increased oxidative stress and cell apoptosis in mesangial cells. BCE and MGF limited mitochondrial membrane potential (ΔΨm) collapse by inhibiting Nox4, mitochondrially bound hexokinase II dissociation, and subsequent ROS production, which effectively reduced oxidative stress, cleaved caspase-3 expression and cell apoptosis. Our work indicated that BCE and MGF had protective effects on diabetic caused kidney injury and prevented oxidative stress in mesangial cells by regulation of hexokinase II binding and Nox4 oxidase signaling.