Background: Oral absorption of peptide-loaded nanoparticles is often limited by multiple barriers of the gastrointestinal epithelium.  Objective: This study aimed to develop a new drug nanocarrier intended to overcome the barriers associated to the oral modality of peptide administration. Materials and Methods: hydrophilic glycocholic acid and hydrophobic deoxycholic acid were chose as targets and a novel nanocomplex constituted of low molecular weight protamine - deoxycholic acid and polysialic acid – polyethylene glycol - glycocholic acid will be developed. The mechanism of transmembrane transport, suborganelle localization and targeting in vitro and in vivo of dual bile acid-modified targeted nanoparticles will be elucidated. Results: The nanocomplexes exhibited a unimodal size distribution with a mean size of 145 nm and a negative zeta potential of -19mV, the capacity to associate exentide (~72% association efficiency) and protect it from degradation in simulated intestinal fluids. In Caco-2 cell monolayers, nanocomplexes are internalized via apical sodium-dependent bile acid transporter (ASBT)-mediated endocytosis. The co-localization of FITC labelled nanoparticles and ASBT were visualized in Caco-2 cells. Despite of this, the accumulation of the exentide-loaded nanocomplexes in the distal ileum could be verified in vivo upon their labeling with Cy7. In the overall long-term pharmacodynamic studys of db/db mice, assessed by blood glucose, hemoglobin A1c, body weight, and blood lipid concentrations, of daily oral nanocomplexes (300 μg/kg) for four weeks were equivalent to or better than daily subcutaneous injections of free exenatide solution (20 μg/kg). Conclusion: Based on these results, we provide a scientific basis for the construction of high-efficiency and low-toxicity bile acid modified nanoparticles for oral delivery of peptide.