Abstract: To investigate the regulatory function of dietary fiber fermentation on intestinal flora, wheat bran dietary fiber was fermented by white-rot fungi and subsequently added into pasta samples in a certain proportion for in vitro gastrointestinal digestion and fermentation. The pH values of the samples and the content of short-chain fatty acids in the metabolites were measured, and the 16S rRNA genes of each samples were sequenced by Miseq high-throughput. The results indicated that fermentation resulted in a significant (P<0.05) increase in protein and soluble dietary fiber (SDF) content in bran dietary fiber compared with unfermented dietary fiber. Both fermented and unfermented bran dietary fibers significantly (P<0.05) reduce intestinal pH values and promote the production of short-chain fatty acids. At the same addition level, the acid-producing capacity of fermented samples was significantly (P<0.05) higher than that of unfermented bran dietary fiber. Both fermented and unfermented bran dietary fibers significantly decreased the diversity of the intestinal flora, and increased the diversity of Lactobacillaceae HT002, Lactobacillaceae and other beneficial bacteria, and reduced the relative abundance of harmful bacteria such as Streptococcus and Escherichia-Shigella. The former exhibited better microbial regulation effects on microorganisms than the latter, increasing the relative abundance of Bacteroidota and decreasing that of Firmicutes. At the same addition level, the Firmicutes/Bacteroidota (F/B) ratio of fermented wheat bran dietary fiber was significantly lower than that of unfermented wheat bran dietary fiber, indicating that fermentation enhanced its ability to regulate intestinal flora. Fermentation can improve the regulatory effects of wheat bran dietary fiber on intestinal microorganisms and short-chain fatty acids, conferring it with higher prebiotic activity.