Black beans are rich in protein which is easily absorbed and has high bioavailability. Black beans grown in selenium rich soil can absorb selenium from the soil and convert it into selenium-enriched black bean polypeptide (SeBSPP). Using selenium enriched yeast (SeYT) pretreatment as a positive control group, the effects of SeBSPP (molecular weight <7kDa, selenium concentration 122mg/kg) on the intestinal barrier, oxidative indexes, benzo[a] pyrene, (B[a]P) metabolism, short chain fatty acids contents and 16S diversity of intestinal flora in mice were analyzed. The results showed that SeBSPP and SeYT both had preventive effects on B[a]P-induced colonic injury in mice, which could increase T-SOD and GSH-Px enzyme activity, reduce H₂O₂ and MDA levels, improve colonic oxidative stress and intestinal SCFAs relative contents. Compared with SeYT, SeBSPP could increase the level of mucin MUC2 in colon tissues, maintain the intestinal enterocytes and repair the intestinal barrier, efficiently reduce the enzymatic activity of AhR, an enzyme related to B[a]P metabolism, inhibit the formation of BPDE-DNA, and reduce toxicity of B[a]P to colon. In addition, SeBSPP could regulate intestinal flora and protect intestinal health by downregulating g_Butyricicoccus and g_Arryflintia, and upregulating g_Lactobacillus, g_Lachnoclostridium and g_Lachnospiraceae_NK4A136_group, etc. This study showed that SeBSPP could effectively prevent B[a]P-induced colon injury by inhibiting B[a]P metabolism and regulating intestinal flora, providing a new approach for the inhibition of B[a]P colon toxicity and the application of selenium polypeptides.