Background & objective: Diphtheria, caused by toxigenic strains of Corynebacterium diphtheria is a fatal disease that was characterized by Hippocrates in 5 B.C. Exotoxin (dTX) is causative agent of this lethal disease. Diphtheria toxin consist two chains, catalytic (A) and binding (B) chain. The toxin binds to its receptor by binding chain (B) and enters in many of body cells such as myocardial, kidney and peripheral nerve cells. After entering, catalytic chain (A) inhibits protein synthesis and cause target cell death. Currently, toxoid form of diphtheria toxin used as vaccine. The aim of this study was design and provide of synthetic mutated catalytic subunit of diphtheria toxin. Then, regarding to its bioinformatics study, this gene would cloned and expressed as recombinant protein vaccine. Methods: After determining and optimizing of dtx gene sequence containing two mutations (A158G, G52E), and perform its bioinformatics study, this synthetic gene was provided in expression vector and molecular analysis was carried out. The pET28a/dtxA construct was transformed in Bl21DE3 E.coli. Then, recombinant protein expression and production as a vaccine candidate was evaluated by western blotting technique. Results: Regarding to the codon optimization of A subunit of diphtheria toxin and bioinformatics analysis, the synthetic mutated gene in expression vector was ordered and provided. The molecular analysis of this gene by restriction digestion and electrophoresis was confirmed its accuracy. This protein was confirmed by western blotting after expression and production. Conclusion: According to advantages of DtxA recombinant protein to diphtheria toxoid, it seems this recombinant protein, as vaccine candidate, could be replaced with diphtheria vaccine. This issue should been completed by more researches.