Previous studies of cellulose nanofibrils (CNF) in decreasing fat absorption and glucose release suggested their potential application as food additives or supplements in diets containing high contents of fat and sugars. However, the long-term effects of CNF uptake remained unknown. The purpose of this study was to determine the effects of subchronic oral CNF consumption on various health aspects of Western diets (WD)-fed mice. The results demonstrated that CNF decreased fat absorption in the jejunum and attenuated WD-induced fatty liver, but slightly decreased lean body mass and affected glucose homeostasis. Additional in vivo studies showed that CNF decreased the intestinal absorption. The in vitro studies suggested that CNF did not decrease the viability of any cells used; however, they prevented epithelial and T cells, but not macrophages, from accessing the viability dye. Taken together, CNF decreased the intestinal absorption non-specifically, which might lead to nutritional risks after long-term exposure.