Abstract Electroporation or electrotransfer is an effective delivery system for introducing plasmid DNA into cells and tissues. During this process, pDNA primarily enters the cell via endocytosis, although some DNA reaches the cytosol. Endosomal, cytosolic, and nuclear DNA-specific pattern recognition receptors (PRRs) in many cell types have been described. Activated PRRs induce the production of proinflammatory molecules and programmed cell death. In B16.F10 mouse melanomas, electrotransfer of empty backbone plasmid induced the production of several proinflammatory cytokines and chemokines including IFNβ, implicating the binding and activation of intracellular DNA-specific PRRs. Histologically, tumor necrosis independent of caspase-3 was observed. The purpose of this study was to investigate whether tumors and cells express cytosolic DNA sensors and whether these sensors respond to pDNA electrotransfer. Although the mRNAs for several DNA sensors were detected in tumors, none was significantly upregulated. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated after DNA electrotransfer. The mRNAs for several DNA sensors were present in these cells and DAI, DDX60, and p204 mRNAs were significantly upregulated after pDNA electrotransfer. DDX60 protein levels were coordinately upregulated. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, increased IFNβ and DNA sensor expression accompanied by cell death and tumor necrosis indicate that DNA electrotransfer activates intracellular DNA sensors in B16.F10 cells and tumors, producing both in vitro and in vivo effects.