Abstract: To investigate the influences of zwitterionic polymer chain length on mucus permeability and cellular uptake, the nanoparticles (NPs) were coated with poly(sulfobetaine methacrylate) (pSBMA) with different chain lengths. The di-block polymer poly(ε-caprolactone)-block-poly(sulfobetaine methacrylate) (PCL-pSBMA) with different chain lengths were synthesized via atom transfer radical polymerization (ATRP) combining with ring-opening polymerization of ε-caprolactone, and corresponding nanoparticles (pSBMAn NPs) were prepared by nanoprecipitation method. The sizes of different pSBMAn NPs were around 100 nm, and zeta potential were about -7 mV. Mucin interaction or mucus penetration study based on transwell systems were employed to evaluate mucus permeability of NPs. Caco-2 cells and mucus-producing HT-MTX-E12 cells were employed to illustrate the endocytosis efficiency of pSBMAn NPs. The results showed that the permeability coefficient of NPs coated with shorter chain length of pSBMA (pSBMA₁₀ NPs) was only 42.83% of that coated with longer pSBMA (pSBMA₈₀ NPs). On the contrary, the cellular uptake of pSBMA₁₀ NPs was 2.44 fold higher compared to pSBMA₈₀ NPs. Although the cellular uptake of pSBMAn NPs was reduced in the presence of mucus, pSBMA₁₀ NPs still presented the highest cellular uptake. However, the in vivo results indicated that the oral bioavailability of pSBMA₂₀ NPs was higher than that of pSBMA₁₀ NPs. All animal procedures were performed in accordance with the Guidelines of the Sichuan University Animal Care and Use Committee and were approved by the Animal Ethics Committee of Sichuan University. This study provides a reference for oral delivery of zwitterionic nanoparticles.