A replication origin of Bacillus thuringiensis (Bt) was found in a Bacillus thuringiensis-Escherichia coli shuttle vector of pHT3101. Deletion analysis showed that the replication origin was segregationally stable at suitable temperature for Bt growth. The fragment containing the replication origin was cloned in pUC18 and sequenced. It was 261 base pairs in length, located in the open reading frame 2 (ORF2) of BTSPB sequence. The 261-bp fragment was cloned in pBR322, creating an improved Bt-E. coli shuttle vector pBR261, which contained two resistance genes responsible for ampicillin and tetracycline. Our study showed that the replication origin structure could be recognized by replication protein of host cells.