ABSTRACTWolbachiamediates antiviral protection in insect hosts and is being developed as a potential biocontrol agent to reduce the spread of insect-vectored viruses. Definition of the molecular mechanism that generates protection is important for understanding the tripartite interaction between host insect,Wolbachia, and virus. Elevated oxidative stress was previously reported for a mosquito line experimentally infected withWolbachia, suggesting that oxidative stress is important forWolbachia-mediated antiviral protection. However,Wolbachiaexperimentally introduced into mosquitoes impacts a range of host fitness traits, some of which are unrelated to antiviral protection. To explore whether elevated oxidative stress is associated with antiviral protection inWolbachia-infected insects, we analyzed oxidative stress of fiveWolbachia-infectedDrosophilalines. In flies infected with protectiveWolbachiastrains, hydrogen peroxide concentrations were 1.25- to 2-fold higher than those in paired fly lines cured ofWolbachiainfection. In contrast, there was no difference in the hydrogen peroxide concentrations in flies infected with nonprotectiveWolbachiastrains compared to flies cured ofWolbachiainfection. Using aDrosophilamutant that produces increased levels of hydrogen peroxide, we investigated whether flies with high levels of endogenous reactive oxygen species had altered responses to virus infection and found that flies with high levels of endogenous hydrogen peroxide were less susceptible to virus-induced mortality. Taken together, these results suggest that elevated oxidative stress correlates withWolbachia-mediated antiviral protection in naturalDrosophilahosts.