The piggyBac (PB) transposon is a recently identified, active and flexible transgene vector, combining the advantages of non-viral gene delivery with genomic integration and persistent transgene expression. In this study, we utilized the PB transposon to carry the herpes simplex thymidine kinase (HSV-tk) and red fluorescent protein (mRFP1) reporter genes into the HeLa cervical cancer cell line or tumor xenografts of cervical cancer. Our data showed that HSV-tk and mRFP1 were expressed in HeLa cells and tumor xenografts three weeks after intratumoral injection. The mRNA and protein levels of HSV-tk and mRFP1 were increased by using the PB transposon vector. Our system also demonstrated that sensitivity of transfected HeLa cells to the pro-drug ganciclovir (GCV) was enhanced in vitro and in vivo. Furthermore, our data indicated that the enhanced transgenic therapeutic effect was strongly associated with high-level transgene expression mediated by the PB transposon. Our results suggest that applying the PB transposon in HSV-tk gene delivery and GCV treatment is a promising gene therapy strategy in the treatment of cervical cancer.