Objectives: The current study was aimed to develop and characterize insulin loaded Chitosan/PLGA microspheres for oral insulin delivery with the objective of avoiding gastric degradation of insulin and improving bioavailability.  Methods: Insulin loaded Chitosan/PLGA microspheres encapsulating insulin were prepared by the emulsification method. The prepared insulin loaded Chitosan/PLGA microspheres were characterized by Scanning electron microscopy (SEM), Fourier transmission infrared (FT-IR) spectroscopy, particle size, zeta potential, entrapment efficiency (EE%), swelling index, in vitro release and stability. In vivo study was done on diabetic Wistar rats. Diabetes was induced by the intravenous administration of alloxan. Blood glucose levels of diabetic rats after oral administration of insulin loaded Chitosan/PLGA microspheres were determined using Glucometer. The histopathology of rat’s colon after administration of FITC loaded formulation was done using confocal microscope. Stability of prepared microspheres was determined by Differential Scanning Calorimetry for 90 days at 4°C ± 1° Results: The optimized formulation was found to have entrapment efficiency of 82.5±2.33% and with mean diameter of 2.0µm. The TEM and SEM photograph of microsphere indicated its spherical shape and smooth surface. The comparison of FTIR spectra of nascent insulin with insulin loaded microspheres indicated that insulin was intact when entrapped in microspheres.  The DSC spectra of formulation stored for 90 days at at 4°C ± 1°C revealed that the formulation was stable.  Conclusions: The in vitro drug release behavior of microspheres were performed in simulated gastric fluid (SGF; pH 2.0) for 2 hours and simulated intestinal fluid (SIF; pH 7.4) from 2 to 40 hours. Plain insulin was 97±2.1In vivo study result revealed that oral administration of insulin loaded Chitosan/PLGA microspheres decrease blood glucose level efficiently within 4 hrs and the consequently these microspheres might be used as potential carrier for oral insulin delivery.