Flavanones in Citrus unshiu peel (CUP) have been used as therapeutic agents to reduce intestinal inflammation; however, the anti-inflammatory effects of their biometabolites remain ambiguous. Here, we identified aglycone-type flavanones, such as hesperetin and naringenin, which were more abundant in the bioconversion of the CUP than in the ethanol extracts of the CUP. We found that the bioconversion of the CUP induced the canonical nuclear factor-κB pathway via degradation of IκB in Caco-2 cells. To check the immune suppressive capacity of the aglycones of the CUP in vivo, we orally administered the bioconversion of the CUP (500 mg/kg) to mice for two weeks prior to the 3% dextran sulfate sodium treatment. The CUP-pretreated group showed improved body weight loss, colon length shortage, and intestinal inflammation than the control mice. We also found a significant decrease in the population of lamina propria Th17 cells in the CUP-pretreated group following dextran sodium sulfate (DSS) treatment and an increase in mRNA levels of occludin in CUP-treated Caco-2 cells. Pyrosequencing analysis revealed a decreased abundance of Alistipes putredinis and an increased abundance of Muribaculum intestinale in the feces of the CUP-pretreated mice compared to those of the control mice. Overall, these findings suggest that the pre-administration of CUP biometabolites may inhibit the development of murine colitis by modulating intestinal permeability and the gut microbiome.