In the present study, we explored and compared the binding activity and immunogenic characterization of the most effective part corresponding to C-terminal quarter of heavy chain of botulinum neurotoxin serotype A (AHc-C) with C-terminal half of heavy chain of botulinum neurotoxin serotype A (AHc). Firstly, the fully soluble AHc-C protein successfully expressed in Escherichia coli by co-expression with thioredoxin (Trx) was shown to bind with ganglioside as the AHc, indicating that the recombinant AHc-C protein retains a functionally active conformation. Furthermore, a solid-phase assay showed that the anti-AHc-C sera effectively inhibited the binding of AHc or AHc-C to the ganglioside GT1b, the first step in BoNT/A intoxication of neurons, as good as the anti-AHc sera. Finally, although the recombinant AHc-C protein still induced a high serum antibody titers and afforded protection level as the mice challenged with active botulinum neurotoxin serotype A, the immunization with AHc protein induced stronger protective potency than the AHc-C protein. The data presented in the report shows that there are the same ganglioside binding activity and different immunogenic characterization between the C-terminal quarter and half of heavy chain of botulinum neurotoxin serotype A. Therefore, the recombinant AHc-C protein can not only be developed into a minimal subunit candidate vaccine for prophylaxis against botulinum neurotoxin serotype A but also be used as a promising tool in the search for binding inhibitors and chimeric vaccines.