It has been reported that the local introduction of a promoter-less DNA containing the complementary DNA (cDNA) sequence of a gene could induce gene-specific silencing in plants. The feasibility of this kind of silencing in human cancer cells is as yet unknown. The current study was designed to investigate the anti-tumor effects of a homologous promoterless K-ras cDNA system on pancreatic cancer. A full-length K-ras cDNA fragment was cloned into the promoterless plasmid puc19 to yield puc-K-ras. This construct was then transfected into pancreatic cancer cells. Our results demonstrated that the transfection of a promoterless K-ras cDNA resulted in a significant decrease in endogenous K-ras in a dose- and time-dependent manner and induced pancreatic cell apoptosis. Furthermore, stable puc-K-ras transfection decreased the endogenous protein level of K-ras and inhibited cell proliferation, clone formation and tumorigenicity in vivo. These findings indicate a promising application of this homologous promoterless cDNA silencing system in pancreatic cancer gene therapy.