RNA interference (RNAi)-based gene therapy has great potential in cancer and infectious disease treatment to correct abnormal up-regulation of gene expression. We show a new original method uses synthetic microRNAs combined with a thermo-inducible promoter to reduce specific gene expression. The targeted gene is the luciferase firefly reporter gene overexpressed in a subcutaneous tumor which allows the RNAi monitoring by bioluminescence imaging (BLI). The inducible inhibition was first demonstrated in vitro using genetically modified cells lines and then in vivo using the corresponding xenograft model in mice. Achieving spatio-temporal control, we demonstrate the feasibility to induce, in vivo, a specific gene inhibition on demand. Future applications of this RNAi-based gene therapy, which can be restricted to pathological tissue, would offer wide-ranging potential for disease treatment.