The botulinum neurotoxins (BoNT) are the most toxic proteins for humans and designated "Category A Select Agents." The current vaccine against botulism is in limited supply, and there is a need to develop new vaccine strategies. A recombinant BoNT/A toxoid was produced in Clostridium botulinum that contained a double amino acid substitution, R363A Y365F (termed BoNT/A(RYM)). BoNT/A(RYM) was noncatalytic for SNAP25 and nontoxic for mice. Immunization with BoNT/A(RYM) protected mice from challenge at levels that were similar to chemically inactivated BoNT/A toxoid. BoNT/A(RYM) elicited an immune response against the light-chain and heavy-chain components of the toxin. Neutralizing anti-BoNT/A(RYM) sera blocked BoNT toxicity in primary cortical neurons and blocked ganglioside binding by the heavy chain. BoNT/A(RYM) represents a viable vaccine candidate for a holotoxoid against botulism.