It is still unclear whether oral administration of mechanically fibrillated cellulose nanofibers (hereafter, fib-CNF), which is widely used among nanofibers, causes toxicity or, on the contrary, improves health, despite the reported safety and even beneficial effects of oral intake of chitin-based nanofibers. Therefore, it is crucial to adhere to the guidelines outlined in OECD TG407 for the evaluation of oral toxicity. This study aimed to assess the safety of orally administered fib-CNF through an acute toxicity study in rats in accordance with the OECD guidelines laid out in TG407 for four weeks. CNF "BiNFi-s" FMa-10005, obtained through the mechanical defibrillation of pulp-derived cellulose, was administered via forced oral administration to male and female Crl:CD(SD) rats at doses of 50, 150, 500, and 1000 mg/kg/day for a period of 28 days in a control group (vehicle: water for injection). The study aimed to assess the toxic effects of repeated administration, and the rats were also monitored for 14 days after the administration to evaluate the recovery from any toxic effects. Results indicate that no deaths occurred in either sex during the administration period, and no toxicological effects related to the test substance were observed in various assessments, including general condition and behavioral function observations, urinalysis, hematological examination, blood biochemical examination, necropsy findings, organ weights, and histopathological examination. Notably, only female rats treated with 1000 mg/kg/day of CNF exhibited a consistent reduction in body weight during the 14-day recovery period after the end of treatment. They also showed a slight decrease in pituitary and liver weights. However, hematological and blood biochemical tests did not reveal significant differences, suggesting a potential weight-suppressive effect of CNF ingestion.