Human mitochondrial Lon is an ATP-powered proteolytic machine that specifically binds to single-stranded G-rich DNA and RNA in vitro. However, it is unknown whether Lon binds mitochondrial DNA (mtDNA) in living cells or functions in mtDNA integrity. Here, we demonstrate that Lon interacts with the mitochondrial genome in cultured cells using mtDNA immunoprecipitation (mIP). Lon associates with sites distributed primarily within one-half of the genome and preferentially with the control region for mtDNA replication and transcription. Bioinformatic analysis of mIP data revealed a G-rich consensus sequence. Consistent with these findings, in vitro experiments showed that the affinity of Lon for single-stranded DNA oligonucleotides correlates with conformity to this consensus. To examine the role of Lon in mtDNA maintenance, cells carrying an inducible short hairpin RNA for Lon depletion were used. In control and Lon-depleted cells, mtDNA copy number was essentially the same in the presence or absence of oxidative stress. However when oxidatively stressed, control cells exhibited an increased frequency of mtDNA lesions, whereas Lon-depleted cells showed little if any mtDNA damage. This suggests that oxidative mtDNA damage is permitted when Lon is present and prevented when Lon is substantially depleted. Upon oxidative stress, mIP showed reduced Lon binding to mtDNA; however binding to the control region was unaffected. It is unlikely that oxidative modification of Lon blocks its ability to bind DNA in vivo as results show that oxidized purified Lon retains sequence-specific DNA binding. Taken together, these results demonstrate that mtDNA binding is a physiological function of Lon and that cellular levels of Lon influence sensitivity to mtDNA damage. These findings suggest roles for Lon in linking protein and mtDNA quality control.