Xylo-oligosaccharide (XOS) is sugar oligomers composed of a β-1,4-linked xylopyranosyl backbone that are obtained by either chemical or, more commonly, enzymatic hydrolysis of xylan polysaccharides extracted from plant cell wall. In this study, acute and subchronic toxicity of XOS in mice and rats have been evaluated, respectively. In the acute study, no obvious clinical signs of toxicity or mortality were observed in mice at the dosage of 32 g/kg BW XOS, excepting transient unformed stools were observed. In the subchronic study, XOS was evaluated in rats with dietary administration at concentrations of 0 (control), 0.9, 2.9, 8.8 and 10% for 13 weeks. Measurements included clinical observations, body weight, food consumption, food conversion efficiency, hematology, blood chemistry, gross necropsy, organ weight and histopathology. Under the conditions, no treatment-related changes were noted in behavior or appearance of the rats and no mortalities occurred. No toxicological findings were found in food consumption, food conversion efficiency, hematology, clinical biochemistry or organ weights in either sex. It is concluded, therefore, that the high dose level, at which the female and male rats consumed about 11.51 and 14.95 g XOS/kg bw/d, respectively, is the no observed adverse effect level (NOAEL) of this 13-week toxicity study.