An attempt was made to prepare and optimize prolonged release nanoparticulate formulation for diltiazem hydrochloride using bioadhesive polymer gelatin by desolvation method, to deliver drug at a controlled rate to its absorption window and thereby improve bioavailability. On the basis of screening studies amount of gelatin and glutarlaldehde were selected for the optimization study, using face centered central composite design (CCD), with α=1. The optimized formulation was evaluated for morphology, stability and X-ray imaging for in-vivo evaluation of the mucoadhesive property. The optimized nanoparticles were found to be discrete and spherical in shape. Accelerated stability studies as per ICH guidelines revealed that there were no significant changes after 6 months. The X-ray studies revealed even after 10 h, NPs were observed to be adhered in the gastric region, since the mucoadhesive property of NPs delay influx at the pylorus. But the barium sulphate suspension (control) was cleared off completely from the stomach within 4 h. The results suggest that the bioavailability of Diltiazem HCl may be enhanced due to the extended retention of mucoadhesive nanoparticles in upper GIT.