AbstractNanoparticles (NPs) used for oral administration have greatly improved drug bioavailability and therapeutic efficacy. Nevertheless, NPs are limited by biological barriers, such as gastrointestinal degradation, mucus barrier, and epithelial barrier. To solve these problems, we developed the PA‐N‐2‐HACC‐Cys NPs loaded with anti‐inflammatory hydrophobic drug curcumin (CUR) (CUR@PA‐N‐2‐HACC‐Cys NPs) by self‐assembled amphiphilic polymer, composed of theN‐2‐Hydroxypropyl trimethyl ammonium chloride chitosan (N‐2‐HACC), hydrophobic palmitic acid (PA), and cysteine (Cys). After oral administration, the CUR@PA‐N‐2‐HACC‐Cys NPs had good stability and sustained release under gastrointestinal conditions, followed by adhering to the intestine to achieve drug mucosal delivery. Additionally, the NPs could penetrate mucus and epithelial barriers to promote cellular uptake. The CUR@PA‐N‐2‐HACC‐Cys NPs could open tight junctions between cells for transepithelial transport while striking a balance between mucus interaction and diffusion through mucus. Notably, the CUR@PA‐N‐2‐HACC‐Cys NPs improved the oral bioavailability of CUR, which remarkably relieved colitis symptoms and promoted mucosal epithelial repair. Our findings proved that the CUR@PA‐N‐2‐HACC‐Cys NPs had excellent biocompatibility, could overcome mucus and epithelial barriers, and had significant application prospects for oral delivery of the hydrophobic drugs.