Abstract: The mannan produced from Saccharomyces has a variety of biological activities. This study aimed to provide basic data of biosafety and distribution for the applications of the yeast mannan in vivo. The mannan bioactivities, including the cytotoxicity to Caco-2 and the hemolysis to red blood cells, were studied in vitro via the MTT method and hemolysis test. In this study, the molecular weight of mannan was 20 kDa. The distribution of the yeast mannan was examined using KM mice administered the solution of yeast mannan with 100 mg/kg BW, then, sugar content was detected over time in the gut of mice (duodenum, jejunum, and ileum), in the blood and the main internal organs (heart, liver, spleen, lung, and kidney). The results showed that the yeast mannan with 20 kDa MW has a great biological safety. The yeast mannan with a concentration of 160 μg/mL showed no obvious toxicity to Caco-2 cells, and exerted a slight promotion effect for cell proliferation rate of 115.6%. In the concentration range of 80-200 μg/mL, the hemolysis of yeast mannan to erythrocytes was less than 5%, which met the national requirements of biosafety. After the mice were gavaged the solution of yeast mannan for 30-90 min, the blood glucose level was significantly increased. The sugar was mainly distributed in the liver and lung during administration for 1 to 3 hours, and the sugar content in the heart and kidney was not changed, while, in the spleen, it decreased slowly. The residues of yeast mannan unabsorbed in the intestine remained in the duodenum for a short time, then the sugar content in jejunum was higher than that in duodenum and ileum at 30 min. After 60 min, the residues of yeast mannan unabsorbed in the intestine flowed to the ileum, yeast mannan was mainly absorbed into the blood in jejunum and ileum during the time of 0 to 90 min. The results of this experiment confirmed that the yeast mannan with 20 kDa MW has high biocompatibility without cytotoxic and hemolytic in vitro, and could be absorbed into the blood in the small intestine, then mainly distributed in the liver and lung in vivo. This study helps to understand the absorption and distribution mechanism of the yeast mannan with 20 kDa MW, provide basic data for the application of yeast mannan in vivo, and give a great prospect for the research and development of polysaccharide drugs as well.