Emerging evidence of the beneficial effects of defatted rice bran (DFRB) on gut health has advanced the development of fermented defatted rice bran as a potential functional food. However, less is known about its effects and underlying mechanisms on gut health. In this study, a mouse model together with fecal microbiota transplantation (FMT) was utilized to study the effects and mechanisms of fermented DFRB (FR) on gut barrier function. We found that FR improved the intestinal morphology, gut tight junction proteins, mucin, antimicrobial peptides, and interleukin 22 (IL-22) and promoted the gut Clostridium butyricum and butyrate. Notably, correlation analysis indicated gut C. butyricum and butyrate were two FR-induced effectors that improved gut health. FMT results suggested that C. butyricum, butyrate, and fecal microbiota from the FR group all reduced prolyl hydroxylase 2 (PHD2) expression by activating peroxisome proliferator-activated receptor gamma (PPARγ) in the mouse colon. This decrease in gut PHD2 subsequently upregulated the hypoxia-inducible factor-1 alpha (HIF-1α) expression, which in turn increased the expression of its targeted downstream tight junction proteins, mucin and antimicrobial peptides, and colonic IL-22 secretion. Overall, FR-derived C. butyricum and butyrate might improve gut barrier function through the HIF-1 signaling pathway, which provides a reference for the application of fermented DFRB as a potential functional food for improving of gut barrier function.