Objective To investigate the subchronic toxicity of yeast β-glucan and to determine its no-observed-adverse-effect-level (NOAEL). Methods Eighty Sprague-Dawley rats were randomly divided into four groups with twenty rats (10 males and 10 females) in each group. Four groups of animals were treated respectively with 0, 0.62，1.25，and 2.50 g/kg BW of test material by gavage for 90 days. Clinical observations were conducted daily. Body weights and food consumption were recorded weekly. Blood samples were collected from all rats on the 45^th day and the day by the end of the study. Gross and histopathologic examinations and organ weights were performed on all animals euthanized for necropsy. Results All parameters including body weight, food consumption, hematology, clinical chemistry, absolute and relative organ weights and histopothological examination in treatment groups were comparable with the control group. Conclusion The NOAEL of yeast β-glucan was 2.50 g/kg BW in male and female rats, which was the highest dose tested in this study and equivalent to 600 times of the quantiy of yeast β-glucan recommended for human use.