Electrotransfer of plasmid DNA in skeletal muscle is a common non-viral delivery method for both therapeutic genes and DNA vaccines. Yet, despite the similar approaches, an immune response is detrimental in gene therapy, but desirable for vaccines. However, the full nature of the immune and tissue responses to nucleic acids and electrotransfer in skeletal muscle has not been addressed. Here we used microarray analysis, fluorescence-activated cell sorting and quantitative polymerase chain reaction to obtain the molecular and cellular signature of the tissue and immune response to electrotransfer of saline and non-coding plasmid DNA. Saline electrotransfer resulted in limited infiltration and induction of a moderate damage-repair gene expression pattern not involving innate immune activation. However, plasmid electrotransfer augmented expression of the same genes in addition to inducing a strong innate immune response associated with pro-inflammatory infiltration. In particular, the inflammasome, Toll-like receptor 9 and other pattern recognition receptors able to respond to cytoplasmic DNA were upregulated. Several key differences in the nature of the inflammatory infiltrate and the kinetics of gene expression were also identified when comparing electrotransfer of conventional and CpG-free plasmids. Our data provide insights into the mechanisms of DNA detection and response in muscle that has relevance for non-viral gene therapy and DNA vaccination.