The aim of this work was to evaluate oral nanocarriers, prepared from zein nanoparticles coated with a poly(anhydride)-thiamine conjugate (GT), for the delivery of insulin. Nanoparticles displayed a size of 250 nm with a negative surface charge, and an insulin loading of 80 mu g/mg. Under simulated gastric conditions, GT-coated nanoparticles released a significantly lower amount of insulin than bare ones; whereas in simulated intestinal conditions, both types of nanoparticles displayed a similar behavior. The effect of insulin on the lipid metabolism of C. elegans under high glucose conditions, characterized by a reduction of the fat content, was also investigated. The effect was significantly higher for the nanoencapsulated forms of insulin than for the free protein (p < 0.001). This effect was two times higher for GT-coated nanoparticles than for bare ones. In rats, the hypoglycemic effect and the pharmacokinetic profile of insulin-loaded nanoparticles orally administered (50 IU/kg) were evaluated. The glycemia of animals slowly decreased reaching a minimum 6-10-h post-administration, with a maximum decrease of about 60%. The pharmacological availability of nanoencapsulated insulin was 13.5%. In serum, nanoparticles provided a maximum of insulin 4-h post-administration, and its relative oral bioavailability was 5.2% (compared with a sc formulation of insulin).