Purpose: Main objective of this study was to develop a novel combination therapeutic strategy for pancreatic cancer, which has a high degree of mortality. Epidermal growth factor receptor (EGFR) targeting peptide-modified thiolated gelatin nanoparticles with encapsulated wild-type p53 expressing plasmid DNA and gemcitabine were administered in Panc-1 human pancreatic adenocarcinoma cells for evaluation of transfection and therapeutic efficacy. Methods: Following synthesis of thiolated gelatin by reacting type B gelatin (pI 4.5 to 5.5) with 2-iminothiolane, the nanoparticles were formed by optimized solvent displacement method. The surface of nanoparticles was modified with heterobifunctional poly(ethylene glycol) (PEG) for attachment of the 12-amino acid EGFR targeting peptide. Reporter and wt-p53 expressing plasmid DNA constructs were encapsulated in the control and EGFR-targeted nanoparticles and administered to Panc-1 cells. Additionally, the nanoparticles were formulated with conjugated gemcitabine. Results: Control (unmodified), PEG-modified, and EGFR targeting peptide-modified thiolated gelatin nanoparticles were prepared with efficient DNA encapsulation and stability. The hydrodynamic diameters of all of these systems were in the range of 150-300 nm. DNA delivery in EGFR-targeted nanoparticles resulted in highest levels of gene expression relative to all the other controls. In the case of p53 plasmid delivery, targeted nanoparticles showed increased level of mRNA transcripts on apoptosis transcription factors. After encapsulation of gemcitabine, EGFR-targeted nanopaticles have significantly decreased IC50 value in cancer cells, compared to gemcitabine itself. Conclusions: The preliminary in vitro results show that EGFR-targeted gelatin nanoparticles can serve as a safe and efficient delivery system for combination gene and drugs therapy for pancreatic cancer.