Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infections an urgent need. We have previously shown that manipulating the lungs? intrinsic host defenses by therapeutic delivery of a unique dyad of inhaled pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. We now report that antimicrobial ROS are therapeutically induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODNs) with mitochondrial voltage-dependent anion channel 1 (VDAC1). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), enhances mitochondrial membrane potential (??m), and differentially modulates ETC complex activities. These combined effects promote leak of electrons from ETC complex III, resulting in antimicrobial superoxide formation. The ODN-induced mitochondrial ROS yield protective effects against an array of pathogens in vitro and in vivo, and the protective ODN effect can be recapitulated through alternate means of VDAC1 manipulation. Together, these studies identify a therapeutic metabolic manipulation strategy that has the potential to broadly protect patients against pneumonia during period of peak vulnerability.