Ulcerative colitis (UC) is a chronic inflammatory disease of the colon that poses significant therapeutic challenges due to the intestinal mucus and epithelial barriers. In this study, ultra-small zwitterionic nanoparticles (HC-CB NPs) is developed based on glutathione (GSH)-responsive hyperbranched polycarbonate to enhance the oral delivery of drugs and overcome these physiological barriers. HC-CB NPs demonstrate high colloidal stability across a wide range of pH environments and physiological fluids, preventing premature drug release within the gastrointestinal tract. The ultra-small sized HC-CB NPs demonstrate minimal mucin adsorption and effectively penetrate through the mucus layer, and the zwitterion surface further facilitate epithelial barrier crossing via the proton-assisted amino acid transporter 1 (PAT1) pathway. HC-CB NPs mediate enhanced macrophage uptake via monocarboxylate transporters (MCTs) pathway and ultimately improved therapy efficacy on colitis. The in vivo results reveal that FK506-loaded HC-CB NPs (HC-CB NPs@FK506) significantly reduce inflammatory markers (TNF-α, IL-6) and myeloperoxidase (MPO) levels, while promoting epithelial integrity by increasing E-cadherin expression. This study offers a promising approach to overcoming intestinal barriers in oral UC treatment, offering biocompatibility and potential for clinical translation. STATEMENT OF SIGNIFICANCE: Ulcerative colitis (UC) is a chronic inflammatory disease of the colon that poses significant therapeutic challenges due to the intestinal mucus and epithelial barriers. This study explores an oral UC therapy using ultra-small zwitterionic nanoparticles (HC-CB NPs) constructed from GSH-responsive hyperbranched polycarbonate. Compared to existing strategies, HC-CB NPs demonstrate minimal mucin adsorption and effectively penetrate through the mucus layer, and the zwitterion surface further facilitate epithelial barrier crossing via the proton-assisted amino acid transporter 1 (PAT1) pathway. Additionally, HC-CB NPs mediate enhanced macrophage uptake via monocarboxylate transporters (MCTs) pathway, resulting in improved therapeutic efficacy. These findings underscore the potential of HC-CB NPs as a transformative platform for overcoming intestinal barriers in UC treatment.