Nanocellulose defines cellulosic compounds smaller than 100 nm in at least one dimension. As a relatively new material, the applications of nanocellulose are expending and there are emerging interests in using nanocellulose as food additives. The mucus layer in the gastrointestinal tract serves as the first defensive barrier to react with foreign objects that enter through the oral route. The interaction between the nanocellulose and mucus gel layer would greatly determine the fate of nanocellulose and reflect the gut health. The goal of the current study was to investigate the behavior of nanocellulose in the GI tract and the health effects, especially the impact on mucus and mucosal layer. Three types of nanocellulose, cellulose nanofiber (CNF), 2,2,6,6-tetramethylpiperidine-1-oxyl radical-oxidized CNF (TEMPO-CNF), and cellulose nanocrystals (CNC), were studied. In the first project, porcine mucosal membrane and mucins were used to investigate mucoadhesion properties of three types of nanocellulose and the mechanisms ex vivo and in vitro. Results showed all nanocellulose possessed mucoadhesive properties in the stomach and the small intestine. In the second part, mice were fed with a Western diet and 30 mg/kg body weight of three types of nanocellulose daily for six weeks. Different nanocellulose did not show a harmful effect on basic physiological parameters. The effect on serum triglycerides, cholesterol and total bile acids and small intestinal morphology was also insignificant. However, the fecal bile output raised for the CNC group. Results suggested that nanocellulose did not improve lipid metabolisms or change intestinal homeostasis at the test dose. In the third part, CNC was tested for its toxicity on cell viability and the translocation across enterocytes. A novel in vitro intestinal mucus model was built to test if the mucus permeability was affected by CNC. It was found that CNC did not permeate through the Caco-2 monolayer or mucus gel layer, nor did it decrease Caco-2 viability. A high concentration of CNC may, however, decrease cholesterol absorption because of the ability of CNC to increase digesta viscosity, decrease mucus permeability, and binding of cholesterol. Our studies provided critical information on nanocellulose on their behavior and functionalities as food additives.