In order to improve the transformation efficiency of the Bacillus thuringiensis (Bt)-Escherichia coli (E. coli) shuttle vector, pHT3101, we intended to minimize replication origin of Bt in pHT3101. For this, two modified shuttle vectors, pHT1K and pHT261, in which 2.9 kb of replication origin of Bt were shortened to 1 kb and 261 bp, respectively as previously reported. Whereas the pHT1K could efficiently transform Bt into the antibiotic resistant, no transformants were obtained with pHT261. Furthermore, pHT1K showed higher transformation efficiency compared to that of parent vector, pHT3101. Therefore, pHT1K might be a very useful Bt-E. coli shuttle vector carrying minimal replication origin of Bt.