Bifidobacterium bifidum has the characteristics of adhering to the intestine and alleviating inflammatory reactions, but the mechanism by which its surface structure functions is not yet clear. In this study, the recombinant expression of the transaldolase (TAL) of Bifidobacterium bifidum E3 (B. bifidum E3), and the TAL protein had adhesion and anti-inflammatory activity. The binding mode of TAL protein to mucins (MUC1 and MUC2) was simulated by molecular docking. Then, the anti-inflammatory effect of TAL protein on IEC-6 cells was investigated in vitro. The TAL protein of B. bifidum E3 was successfully expressed in Escherichia coli M15. Simulation results showed that TAL protein mainly was bound to MUC1 and MUC2 proteins through hydrogen bonding forces. Studies on the anti-inflammatory activity of TAL protein indicated that it was non-toxic to IEC-6 cells. The protein had an alleviating effect on TNF-α-induced inflammation in IEC-6 cells by promoting cell proliferation, reducing apoptosis, regulating cytokine balance and signaling pathways related to intestinal inflammation. Further, TAL protein reduced the nuclear translocation of the p65 subunit in the NF-κB signaling pathway and downregulated the expression of the NF-κB signaling pathway related genes p65 and p50, and upregulated the expression of the PI3K/AKT signaling pathway related genes PI3K and AKT, at both gene and protein levels. Therefore, based on the above results, it was indicated that the transaldolase of B. bifidum E3 bound to mucin receptors and had anti-inflammatory properties, providing a theoretical basis for the development of Bifidobacterium surface proteins as bioactive substances.