The microspheres of Isabgol husk were prepared by emulsification-crosslinking technique and the gastrointestinal transition behavior of the formulation was studied by gamma scintigraphy. The impact of different process variables such as amount of glutaraldehyde, concentration of Isabgol husk and temperature was studied on surface morphology and mucoadhesion. In vitro mucoadhesive testing of formulations was performed by determination of zeta potential, mucus glycoprotein assay and mucus adsorption isotherms. The effect of feeding on retention of microspheres in the gastrointestinal track (GIT) was studied in albino rabbits by gamma scintigraphy study. The results indicated the formation of microspheres as observed by scanning electron microscopy. The smooth and round surfaces of microspheres were obtained on increasing Isabgol husk and glutaraldehyde amount. The positive zeta potential of all formulations indicated the electrostatic interaction as a mechanism of mucoadhesion between the mucus of GIT membranes and the microspheres surfaces. The influence of electrostatic interaction on mucoadhesion of microspheres was again ascertained when the mucin equilibrium adsorption on preparations indicated well fitness in Langmuir and Freundlich adsorption isotherms. During gamma scintigraphy, the stability of (99m)Tc-sodium pertechnetate was found 98.82% at pH 6.8 and 96.78% at pH 7.2, respectively. It indicated the minimal leaching of bound radionuclide from microspheres during gastrointestinal transition as observed in gamma scintigraphic images of the rabbits. The microspheres retained in GIT even after 24 hrs of oral administration. The results indicated the applicability of Isabgol husk mucilage in the development of mucoadhesive microspheres.