To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157: H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B ( stx2b ) from EHEC O157: H7chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokaryotic expression plasmid pET-28a ( + )-eaeC300, which was constructed previously. The recombinant pasmid pET-28a( + )-stx2b-eaeC300 was transformed into E. coli BL21 (DE3). After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Weste blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophylactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with Al(OH)3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC O157:H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-IntiminC300 was successfully cloned into pET-28a (+) vector. The results of SDS-PAGE and Weste blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25% of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75% after primary purification. Animal tests revealed that the fusion protein Stx2B-IntiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophylactic potential.