Background: Intestinal barrier injury contributes to multiple diseases such as obesity and diabetes, whereas no treatment options are available.Methods: Due to close interactions between mucosal microbiome and intestinal barrier, we evaluated the potential of mucosal bacteria in providing targets for high‐fat diet (HFD)–related intestinal barrier injury. Whole‐genome metagenomics was used to evaluate mucosal microbiome, while intestinal barrier injury was estimated using serum LPS, FITC‐dextran intensity, and ZO‐1 protein.Results: We found that HFD induced significant fat accumulation in epididymal tissue at weeks 4 and 12, while ALT, LDL, and TC increased at week 12. Intestinal barrier injury was confirmed by elevated serum LPS at both weeks, upregulated FITC‐dextran intensity, and decreased ZO‐1 protein at week 12. Fourteen species such as Phocaeicola vulgatus differed in HFD‐fed mice. The co‐occurrence network of mucosal microbiome shifted from scale‐free graph in controls to nearly random graph in HFD‐fed mice. Besides, 10 hub bacteria especially Bacteroides ovatus decreased drastically in both mucosal and fecal samples of HFD‐fed mice, correlated with intestinal permeability, ALT, and KEGG pathways such as “Mitochondrial biogenesis” and “metabolism”. Moreover, Bacteroides ovatus has been confirmed to improve intestinal barrier function in a recent study.Conclusion: Mucosal hub bacteria can provide potential targets for improving HFD‐related intestinal barrier function.