Drug repositioning, the repurposing of existing drugs for new applications, provides high safety but is still limited in gene therapy. As gene therapy research grows globally, safe gene manipulation technologies are increasingly needed. Cre recombinase, used in the Cre-loxP system to remove pathogenic genes, is difficult to control temporally. Previous drug repositioning strategies for Cre ON/OFF control showed low DNA recombination efficiency, forming a barrier to medical use. In this study, a gene manipulation technology was developed in which Cre activity is temporarily inactivated (OFF state) and reactivated (ON state) by the repositioned drug trimethoprim (TMP). A dimerization motif, the leucine zipper, was added to enhance ON-state activity, achieving an eightfold increase in DNA recombination efficiency compared with previous methods.