Nanoparticles (NPs) have shown a certain potential to overcome the drawbacks of oral peptide delivery in the gastrointestinal tract such as low peptide stability and permeability. The preparation of insulin loaded NPs was carried out with Eudragit RL or RS dissolved in different non-toxic polyethylene glycol (PEG) derivatives. The use of these non-toxic solvents allowed the design of an one step NP preparation method where insulin retained its full biological activity as it was proven in vitro and in vivo. The insulin trapping NPs were in a size range of around 150-250 nm and exhibited a pH-dependent release. The type of solvent did not distinctly influence the particle properties or insulin stability but modified significantly the performance in vivo in rats, NPs prepared with glycofurol led to a bioavailability of F=1.4 ± 1.0% after oral administration while NPs prepared with PEG 300 were hardly efficient (F=0.3 ± 0.5%). In all cases t(max) was shifted to 2h compared to 1h after subcutaneous insulin solution. In general, we believe that the method presented here is a promising way to encapsulate sensitive drugs, especially for the production of peptide loaded NPs.