In this study, the effect of drug loading on the nasal absorption of insulin was determined. Human insulin was loaded into different drug carriers by two methods: supercritical fluid processing and freeze-drying. The powder formulations were characterized and then evaluated after nasal administration to alloxan induced diabetic rabbits at a dose of 5U/kg and 7.5U/kg. The blood glucose levels and serum insulin levels were monitored for five hours after administration of insulin formulations. The drug carriers evaluated were: ammonium glycyrrhizinate (AG), polyacrylic acid (PAA), crosslinked polyacrylic acid (CPAA), polyethylene oxide (PEO) and chitosan (CHTN). Nasal administration of AG infused with insulin by carbon dioxide resulted in absolute bioavailability of 9.81% as compared to 2.86% observed with same powder loaded with insulin by freeze-drying. 8.05% bioavailability was obtained with PAA powder loaded with insulin by carbon dioxide as compared to much lower absorption seen with freeze-dried formulation. Similarly a two fold increase in absolute bioavailability was observed when carbon dioxide infused CPAA powder formulation was compared to the lyophilized powder. Nasal administration of PEO and CHTN loaded with insulin by carbon dioxide resulted in bioavailabilities of 1.55% and 1.18% respectively. The drug-loading process seems to have a significant effect on nasal absorption of insulin. The powders loaded with insulin by carbon dioxide infusion resulted in significantly higher absorption. The exact mechanism is still not known and a possible explanation for increased absorption may be due to improved stability of insulin in carbon dioxide infused formulations. Among the powders evaluated, polyacrylic acid and ammonium glycyrrhizinate prepared by carbon dioxide infusion as drag-loading method seem to offer good potential for development of nasal powder dosage forms for insulin.