Abstract: To improve the oral bioavailability of insulin, an insulin-loaded enteric polymer-lipid hybrid nanoparticles(INS-NPs L100)was prepared using methoxy PEG-poly(D, L-lactide)(PEG-PLA), phospholipid s75 and Eudragit L100; in vitro and in vivo behaviors of INS-NPs L100 were evaluated. Insulin-loaded polymer-lipid hybrid nanoparticles(INS-NPs)were prepared by W/O/W double emulsion solvent evaporation method. INS-NPs formulation was optimized by single factor experiment using encapsulation efficiency, particle size, and in vitro release behavior of the corresponding INS-NPs L100 as evaluation indexes. The morphology, in vitro drug release profile and hypoglycemic effect of the INS-NPs L100 using the optimal INS-NPs and Eudragit^® L100(used as enteric polymer)were assessed. The results showed that the encapsulation efficiency of the optimal INS-NPs was(62. 18±4. 51)%. The average particle size, PDI and Zeta potential was(225. 2±94. 3)nm, 0. 191±0. 068, and -(14. 84±1. 26)mV, respectively. The cumulative drug release from the INS-NPs L100 was only 8. 01% at 2 h in pH 1. 0 HCl solution, exhibiting a slow drug release behavior; while the drug release from INS-NPs L100 was 67. 31% at 6 h in phosphate buffer of pH 6. 8. Mereorer, after oral administration of INS-NPs L100 with a dose of 38 IU/kg, the blood glucose concentration of healthy rats was reduced to 76% of the initial values at 3. 5 h, exhibiting a sustained hypoglycemic effect. In summary, the INS-NPs L100 prepared in this study could effectively decrease the release rate of insulin in gastric juice, improve the stability of protein in the gastrointestinal tract, and provide a new approach for the oral administration of peptides and protein drugs.