AbstractTherapeutic manipulation of the gasotransmitter hydrogen sulfide (H₂S) has recently been proposed as a novel targeted anticancer approach. Here we show that human lung adenocarcinoma tissue expresses high levels of hydrogen sulfide (H₂S) producing enzymes, namely, cystathionine beta-synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), in comparison to adjacent lung tissue. In cultured lung adenocarcinoma but not in normal lung epithelial cells elevated H₂S stimulates mitochondrial DNA repair through sulfhydration of EXOG, which, in turn, promotes mitochondrial DNA repair complex assembly, thereby enhancing mitochondrial DNA repair capacity. In addition, inhibition of H₂S-producing enzymes suppresses critical bioenergetics parameters in lung adenocarcinoma cells. Together, inhibition of H₂S-producing enzymes sensitize lung adenocarcinoma cells to chemotherapeutic agents via induction of mitochondrial dysfunction as shown in in vitro and in vivo models, suggesting a novel mechanism to overcome tumor chemoresistance.