To overcome gene therapy barriers such as low transfection efficiency and nonspecific delivery, liposomal nanoparticles targeted by a single-chain antibody fragment to the transferrin receptor (TfRscFv) delivering wild-type (wt) human p53 (SGT-53) were developed for tumor-specific targeting. We hypothesize that SGT-53 in combination with gemcitabine will demonstrate enhanced therapeutic benefit in an in vivo metastatic pancreatic cancer model. Intrasplenic injection of 1 × 106 Panc02 murine pancreatic cancer cells was used to generate in vivo hepatic metastatic tumors. Nanoparticle localization was assessed by tail vein injection of TfRscFv with fluorescently labeled oligonucleotides (6-carboxyfluorescein phosphoramidite (6FAM) ODN) imaged by Xenogen IVIS 200 scan. SGT-53 (equivalent to 30 μg of p53 intravenously) and gemcitabine (20 mg/kg intraperitoneally) alone and in combination were administered biweekly and compared with untreated mice. Survival was determined by blinded daily assessment of morbidity. Human wtp53 expression and transferrin levels in the tumors were assessed by western blot analysis. Tumor burden was quantified by liver weight. Xenogen imaging demonstrated tumor-specific uptake of TfRscFv-6FAM ODN. Exogenous human wtp53 protein was detected in the SGT-53-treated tumors compared with control. Compared with untreated mice with metastatic tumors demonstrating median survival of 20 days, SGT-53, gemcitabine and the combination demonstrated improved median survival of 29, 30 and 37 days, respectively. The combination treatment prolonged median survival when compared with single drug treatment and decreased tumor burden. The tumor targeting liposomal-based SGT-53 nanoparticle is capable of sensitizing pancreatic cancer to conventional chemotherapy in pancreatic cancer models. This approach has the potential to be translated into a new, more effective therapy for pancreatic cancer. Further optimization is ongoing, moving towards a Phase 1B/2 clinical trial.