In this study， the carotenoid produced by Neurospora spore was fermentated in vitro after digestion. Carotenoid was extracted from conidial power by acid-temperature method. The fermentation liquid was determined by national standard method of pH， gas production， NH3-N， gas chromatography was used to measure the short chain fatty acid of fermentation broth， the effect of carotenoid on intestinal flora was obtained by 16SrRNA high-throughput sequencing technology. The result showed that after in vitro fermentation， the pH value of fermentation liquid was 6.0-7.2 which was close to the normal pH value of human instestine. The fermentative solution produced litter gas， and much short-chain fatty such as acrtic acid， propionic acid and butyric acid. Moreover， fermentation liquid could reduce bacteroidetes while increased proteobacteria. To sum up， the carotenoid produced by Neurospora spore is beneficial to improve the human intestinal flora， optimize the proportion of bacteria in the intestinal flora， beneficial to the human intestinal health.