The pRNA (packaging RNA) of bacteriophage phi29 DNA packaging motor has been reported to have novel applications in nanotechnology and nanomedicine. The unique ability of pRNA to form dimers, trimers, hexamers and patterned superstructures via the interaction of two reengineered interlocking loops makes it a promising polyvalent vehicle to load siRNA and other therapeutic molecules and be applied as a therapeutic nanoparticle in tumor therapy. In this study, several tumor cell lines were used to evaluate the previously reported pRNA nanotechnology for specific siRNA delivery and for the silencing of targeted gene. It was found that MCF-7 and HeLa cells, out of twenty-five tested tumor cell lines, expressed high levels of folate receptors and exhibited specific binding of the FITC-folate-pRNA nanoparticles, while the others expressed low levels and thus, for these, delivery was not feasible using folate as a targeting agent. Folate receptor positive tumor cells were then incubated with the chimeric pRNA dimer harboring both the folate-pRNA and the chimeric pRNA/siRNA (survivin). Knock down effects of survivin expression in these tumor cells were detected at the mRNA level by real time-PCR and at the protein level by western blot. Apoptosis was detected by flow cytometry analysis with dual staining of annexinV-FITC and PI. The data suggest that the chimeric pRNA nanoparticles containing folate-pRNA and pRNA/siRNA (survivin) could be specifically taken up by tumor cells through folate receptor-mediated endocytosis, resulting in significant inhibition of both transcription and expression of survivin in tumor cells and triggering cell apoptosis. Using such protein-free nanoparticles as therapeutic reagents would not only allow specific delivery and extend the in vivo retaining time but also allow long-term administration of therapeutic particles, therefore avoiding the induction of antibodies caused by repeated treatment for chronic diseases.