Anuroctoxin gene was amplified from source plasmid and cloned into pPICZαA vector using the restriction ligation approach. The recombinant vector was transformed into bacterial strain E. coli using heat shock method. Finally, the correct cloning of gene into desired vector was authenticated by DNA sequencing. In conclusion, I successfully constructed pPICZαA-AnTx vector for the expression of Anuroctoxin in Pichia pastoris expression system.