Delivery of DNA encoding therapeutic genes in vivo has great potential for treating malignancy as well as genetic diseases. Delivery of placebo DNA without a transgene is used as a control in gene therapy studies. It is tacitly assumed by most investigators that the protein expressed from the transfected DNA has phenotypic consequences, but that the consequences are not from the DNA itself. Here, we demonstrate that transfection of control plasmid DNA (that does not express a gene product) into tumor cell lines induces a dramatic (>10-fold) increase in the expression of the interferon (IFN)-regulated genes IRF7, STAT1, MIG (approved gene symbol CXCL9), MHCI (MICA), and CD11a (ITGAL) in tumor cell lines. Induction of these genes inhibits tumor development and tumor growth in immunocompetent mice that are immunized with apoptotic tumor cells. The antibody depletion study indicates that the underlying mechanism by which transfection of control DNA induces IFN-regulated genes is the induction of a secreting factor(s) such as IFN-β. Three lines of evidence indicate that DNA transfection-mediated induction of IFN-regulatory genes is independent of TLR9. The three lines of evidence are: (1) TLR9 is not expressed in either SCCVII or 4T1 cell line, (2) activation of TLR9 downstream signaling molecules is not associated with the induction of gene expression, and (3) the secretion factor(s) obtained from the conditioned medium of DNA-transfected SCCVII tumor cells induces the same type of gene expression in the 4T1 tumor cell line, which is refractory to the gene induction by DNA transfection. Our finding indicates that the 4T1 tumor cell line, which is resistant to the DNA transfection-mediated induction of IFN-regulated genes, can be used to determine the real therapeutic gene function.