Secretion of tumor necrosis factor-α (TNF-α) by macrophages plays a predominant role in the development and progression of various inflammatory diseases. In the current contribution, multifunctional nanoparticles (NPs) containing TNF-α siRNA targeting macrophages via oral administration were developed to knockdown TNF-α expression against acute hepatic injury in rats. Mannose-modified trimethyl chitosan-cysteine (MTC) NPs were prepared by self-assembly method (sa-MTC NPs), ionic gelation and siRNA entrapment method (en-MTC NPs), and ionic gelation and siRNA adsorption method (ad-MTC NPs). Among them, en-MTC NPs demonstrated the best stability against ionic challenges with desired siRNA integrity against nucleases. By targeting normal enterocytes and M cells that express mannose receptors, en-MTC NPs notably promoted intestinal absorption of siRNA in rats. They further facilitated siRNA internalization by rat peritoneal exudate cells (PECs) via lipid-raft involved endocytosis and macropinocytosis, thus inducing effective in vitro TNF-α knockdown. Orally delivered en-MTC NPs at a low siRNA dose of 50 μg/kg inhibited systemic TNF-α production and decreased TNF-α mRNA levels in macrophage-enriched liver, spleen, and lung tissues, which consequently protected rats from acute hepatic injury. Therefore, the en-MTC NPs would provide an effective approach to orally deliver TNF-α siRNA for the anti-inflammatory therapy.