Tetracycline-inducible gene expression (Tet-on) system is a particularly powerful tool for transgenic research and has become one of the first choices for the control of transgene expression in a mammal and a fly. Previously, we have generated the modified reverse tetracycline-controlled transactivators and tetO promoters for a Bombyx mori Tet-on system. In order to further improve this system, Giant, a transcriptional silencer from Drosophila melanogaster, is introduced to repress leaky transcription in the absence of doxycycline. Further, the promoter responsibility to the tetracycline-controlled transactivators is facilitated by introducing a synthetic minimal core promoter. With the tightly regulated second-generation silkworm Tet-on system, we obtain up to 300-fold induction of gene expression with the addition of doxycycline.