Aims: To increase the Cry1Da production in Bacillus thuringiensis by enhancing BtI promoter activity and fusion with upstream sequence from cry1Ab. Methods and Results: The effects of joining the upstream sequence of cry1Ab that contains E2 subunit pyruvate dehydrogenase (PDH) recognition site to the cry1Da promoter as well as the effects of substitution mutation of conserved sequences of its BtI promoter on cry1Da expression was monitored by constructing cry1Da promoter-lacZ fusions. Changing the -35 region of the cry1Da BtI promoter to that of cry1Ab enhanced beta-galactosidase activity about three fold as comparing to that of the wild-type promoter with its own upstream sequence. In contrast, the same cry1Da mutated promoter linked to the above upstream sequence of cry1Ab enhanced enzyme activity up to seven fold, but was five fold lower than that of the full-length cry1Ab promoter. The cry1Ab-cry1Da hybrid promoter with the -35 BtI mutation efficiently increased Cry1Da synthesis by 133% and resulted in a 2.3-fold increase in insect larval toxicity when comparing to the wild type. Conclusions: The cry1Ab promoter as well as mutation of -35 region of BtI promoter together with fusion with E2 subunit PDH recognition site efficiently enhanced Cry1Da production in B.thuringiensis. Significance and Impact of the study: The results provide useful information to construct an efficient cry1Da gene expression in B.thuringiensis.