This study aimed to develop cyclosporine A (CsA)-loaded mucopenetrating nanoparticles (MNP) for the safe and effective treatment of inflammatory bowel diseases (IBD). Poly(ethylene glycol)-block-poly(lactic-co-glycolic acid) (PEG-PLGA) was selected as the mucopenetrating carrier polymer for CsA/MNP to achieve topical accumulation by passive targeting and prolonged pharmacological action at the inflammation site, potentially maximizing anti-inflammatory effects with minimizing systemic exposure. Uniform nanoparticles were successfully prepared using a flash nanoprecipitation technique, and their size and dispersibility varied based on the length of the PEG chain and mixing ratio of PEG-PLGA and PLGA. The composition of CsA/MNP was optimized in terms of the dispersibility and release properties. CsA/MNP exhibited a 3-fold higher penetrating amount through artificial mucus than the reference PLGA nanoparticles. In a chemical-induced rat model of IBD, CsA/MNP accumulated at the inflamed colon site, achieving a 4-fold higher concentration of CsA than the reference PLGA nanoparticles. After repeated oral administration of CsA/MNP, significant anti-inflammatory effects were observed in the model rats compared to the reference formulation as evidenced by 73% reduction of myeloperoxidase activity in the colon tissue of the IBD model animal. Systemic exposure of CsA after a single oral administration of CsA/MNP was 25% of that after oral administration at the toxic dose of CsA. Even after repeated oral administration, no significant changes were observed in plasma biomarkers for kidney functions. PEG-PLGA-based nanoparticles may be viable oral drug delivery carriers to enhance the therapeutic potential of CsA with reduced risk of systemic side effects.