The intestinal epithelial and mucus barriers on the gastrointestinal tract limit the bioavailability of oral protein or peptide drugs. Therefore, efficient mucus permeability and cellular internalization are required properties for oral delivery systems. To overcome these two obstacles, porous silicon nanoparticles were modified with poly (pyridyl disulfide ethylene phosphate/sulfobetaine) polymers to make P(PyEP-g-SBm)n-AmPSiNPs (m = 0.1, 0.2, 0.3 and n = 10, 20, 30) nanoparticles (NPs). The insulin-loaded P(PyEP-g-SB)-AmPSiNPs showed favorable stability and good biocompatibility in vitro. The zwitterionic dodecyl sulfobetaine (SB) coated nanoparticles improved the mucus permeability. P(PyEP-g-SBm)20 with the optimal conjugated ratio (m = 0.3) of SB units was determined by evaluating the mucus diffusion rate of NPs. The cellular uptake of P(PyEP-g-SB0.3)n-AmPSiNPs (n = 10, 20, 30) was much higher than AmPSiNPs in the presence of inhibitors (N-acetylcysteine solution and sodium chlorate) (p < 0.01) due to the enhanced charge shielding effect of P(PyEP-g-SB) modification. The P(PyEP-g-SB0.3)20-AmPSiNPs showed about 1.4-1.7 fold increase in the apparent permeability of insulin across Caco-2/HT-29-MTX cell monolayers, compared to AmPSiNPs (p < 0.01). Finally, the in vivo study showed that insulin-loaded P(PyEP-g-SB0.3)20-AmPSiNPs generated 20% reduction of the blood glucose level with an 2-fold increase in oral bioavailability. These suggested that zwitterionic polyphosphoester modified porous silicon nanoparticles, which were of enhanced mucus permeability and cellular internalization, represent a promising carrier for oral delivery of peptide and protein.