Polymeric nanoparticles are widely used as targeted carriers for biomacromolecules. In this paper, modified gelatin nanoparticles were prepared and their feasibility as insulin pulmonary administration system was investigated. D: ,L: -glyceraldehyde and poloxamer 188 were used for gelatin nanoparticle preparation. Novel water-in-water emulsion technique was used to prepare insulin-loaded nanoparticles. Morphological examination of insulin-loaded nanoparticles was carried out using scanning electron microscopy (SEM). Intratracheal instillation of insulin-loaded nanoparticles was performed to evaluate animal hypoglycemic effect. With fluorescence labeling of insulin, alveolar deposition and absorption of insulin-loaded nanoparticles were investigated. Histological changes in the lung were also observed to evaluate the safety. From the micromorphology observation, insulin-loaded nanoparticles under gelatin-poloxamer 188 ratio at 1:1 showed smooth and uniform surface, with average particle size 250 nm and Zeta potential -21.1 mV. From animal experiment, insulin-loaded nanoparticles under gelatin-poloxamer 188 ratio at 1:1 promoted insulin pulmonary absorption effectively and showed good relative pharmacological bioavailability. Proved by alveolar deposition result, FITC-insulin-loaded nanoparticle group was characterized by an acute and rapid hypoglycemic effect. In addition, nanoparticles could guarantee the safety of lung by reducing insulin deposition in lung. A transient weak inflammatory response was observed at 1 day after administration. With good physical characterization, high bioavailability, fast and stable hypoglycemic effect, insulin-loaded nanoparticles might be developed as a novel insulin pulmonary system for diabetes therapy.