The formation of protein corona (PC) is important for promoting the in vivo delivery of nanoparticles (NPs). However, PC formed in the physiological environment of oral delivery is poorly understood. Here, we engineered seven types of trimethyl chitosan-cysteine (TC) NPs, with distinct molecular weights, quaternization degrees, and thiolation degrees, to deeply investigate the influence of various PC formed in the physiological environment of oral delivery on in vivo gene delivery of polymeric NPs, further constructing the relationship between the surface characteristics of NPs and the efficacy of oral gene delivery. Our findings reveal that TC7 NPs, with high molecular weight, moderate quaternization, and high sulfhydryl content, modulate PC formation in the gastrointestinal tract, thereby reducing particle size and promoting oral delivery of gene loaded TC7 NPs. Orally delivered TC7 NPs target macrophages by in situ adsorption of apolipoprotein (Apo) B48 in intestinal tissue, leading to the improved in vivo antihepatoma efficacy via the natural tumor homing ability of macrophages. Our results suggest that efficient oral delivery of genes can be achieved through an in situ customized ApoB48-enriched PC, offering a promising modality in treating macrophage-related diseases.