Previously, we have shown that chickens immunized with Shiga toxin (Stx) produce Stx-neutralizing egg yolk immunoglobulin Y (IgY) antibody. The anti-Stx-1 IgY and anti-Stx-2 IgY exert their neutralizing activity through their antibody activity against the B subunit of the toxin but not the A subunit. In the present study, chickens were immunized with recombinant Stx-1 B subunit (rStx-1B) and recombinant Stx-2 B subunit (rStx-2B). Induced anti-rStx-1B and anti-rStx-2B IgY neutralized the toxicity of Stx-1 and Stx-2 against HeLa 229 cells. The neutralizing activity of anti-rStx-1B IgY on Stx-1 was almost 10 times stronger than that of anti-Stx-1 IgY, and that of anti-rStx-2B IgY was 2.6 times stronger than that of anti-Stx-2 IgY. Anti-rStx-1B and anti-rStx-2B IgY reacted with multimeric and monomeric forms of the B subunits in contrast to anti-Stx-1 and anti-Stx-2 IgY that reacted with only the multimeric form. These results indicated that recombinant B subunits were promising antigens for induction of neutralizing antibodies in chickens.