Fhit protein is lost in most cancers, its restoration triggers apoptosis and FHIT viral-mediated gene therapy suppresses tumors in preclinical models. We have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that likely mediate Fhit import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from Nadph to cytochrome P450 via ferredoxin. Fhit restoration increases production of reactive oxygen species and the rate of apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.