Nasal delivery Of insulin is an alternative route for administration of this drug. The objective of this study was preparation of chitosan microspheres for insulin nasal delivery. After preparation of insulin chitosan microspheres by emulsification-cross linking process, the effect of chitosan quantity (200-400 mg), cross-linker type (ascorbic acid or ascorbyl palmitate) and amount (70-140 mg) were studied on the morphology, particle size, loading efficiency, flow and release of insulin from the microspheres by a factorial design. Optimized formulation was administered nasally in four groups of diabetic rats and their serum insulin levels were analysed by the insulin enzyme immunoassay kit and the serum glucose by the glucose oxidase kits. Insulin loading in microspheres was between 4.7-6.4% w/w, preparation efficiency more than 65% and mean particle size was 20-45 mum. In most cases, drug released followed a Higuchi model. Ascorbic acid caused an increase in stability. particle size and T-50% while decreased the loading efficiency and production efficiency. Increasing the chitosan content, increased particle size, How and insulin release rate form the microspheres. The increase of cross-linking percentage decreased the flow and size of the microspheres while increase of cross-linking percentage promoted the stability and decreased DES% of insulin. Microspheres containing 400 mg of chitosan and 70 mg ascorbyl palmitate caused a 67% reduction of blood glucose compared to i.v. route and absolute bioavaliability of insulin was 44%. The results showed that chitosan microspheres of insulin are absorbable from nasal route.