A niosomal formulation for simultaneous encapsulation and release of hydrophobic and hydrophilic antidiabetic drugs is reported. The niosomes were prepared by thin film hydration method which involved self assembly of Tween 80 and cholesterol. Confocal laser scanning microscopy (CLSM) investigations revealed that nile red (the hydrophobic molecule) preferentially accumulated in the shell while FITC-dextran (the hydrophilic molecule) loaded in the aqueous core of the niosome. Fourier transform infra-red (FTIR) spectroscopy investigation of niosomes revealed hydrogen bond formation between tween 80 and cholesterol. The encapsulation efficiencies for glipizide and metformin hydrochloride (metformin HCl) were found to be 67.64% and 58.72%, respectively. The drug release experiments performed in buffers at different pH values (mimicking the blood plasma pH, cellular endosomal and gastric environments) revealed that the drug release followed a linear profile up to 8-10 h, slowed down, and lasted for 12-14 h. Thus, this formulation offers a promising drug delivery system for combinatorial sustained release of anti-diabetic drugs. (C) 2017 Elsevier B.V. All rights reserved.