1,5-anhydro-d-fructose (1,5-AF) is a novel monosaccharide produced by the action of alpha-1,4-glucan lyase (EC 4.2.2.13) on glycogen, starch, or related substrates such as maltose and maltosaccharides. 1,5-AF is of interest as a compound to be used as a food supplement because of its antioxidant, antimicrobial, and antidiabetic properties. This enforces the safety of 1,5-AF and therefore, in the current study, four groups of male and female Sprague-Dawley rats were provided with 1,5-AF in the drinking water (at 0 or 1.0 g/kg body weight daily) for a period of 90 days (n=10 in each group). All the animals survived, and no clinical signs of toxicity or alterations in hematological or clinical chemistry parameters were observed. Furthermore, organ weight and histopathological examination of brain, heart, urinary bladder, gastrointestinal tract, and pancreas were normal after 1,5-AF treatment. Moreover, there was no change in food consumption, water intake, or body weight gain in rats receiving 1,5-AF. In conclusion, administration of 1,5-AF did not induce any significant toxicological effects and, therefore, 1,5-AF seems safe to administer in vivo over a long period of time.