Vitellogenin (Vg) has been shown to be involved in host immune defense. However, the underlying mechanism by which Vg functions is largely unknown, and which component in Vg is essential for the execution of its immune role remains lacking. Here, we demonstrate clearly that fish Vg is capable of killing the whole cells of Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus rather than their protoplasts; and that Vg has distinct binding sites specific for lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN), respectively. Of note, the interaction between Vg and bacterial cells via the different binding sites results in distinct effects: the binding of Vg to E. coli via LPS and to S. aureus via LTA is lethal, whereas the binding of Vg to S. aureus via PGN is not. Moreover, Vg exhibits a lectin-like activity because its antibacterial activities can be suppressed by the carbohydrates like d-mannose, N-acetyl-d-glucosamine and d-fucose. Finally, the polypeptide chain integrity and carbohydrate residues of Vg are indispensable for its antibacterial activity, but the lipidation and phosphorylation are not necessary. Taken together, Vg is a bacteriocidal factor capable of killing E. coli and S. aureus whole cells via interaction with LPS and LTA existing in the bacterial cell walls rather than attacking their plasma membranes.