Significance Wolbachia ( w Mel strain)-infected Aedes aegypti mosquitoes are refractory to disseminated arboviral infections. Yet previous studies into the mechanism behind Wolbachia -mediated virus blocking have not considered the involvement of lipids, apart from cholesterol, during superinfection. We used liquid chromatography mass spectrometry to study the lipidome in mosquito cells infected with virus, w Mel, or superinfected with both virus and w Mel. Interestingly, a class of lipids, acyl-carnitines increased during virus infection but remained low with w Mel. These findings uncover a previously unknown role for acyl-carnitines in the interaction among virus, w Mel, and cells, suggesting a mechanism underlying Wolbachia -mediated pathogen blocking. Importantly, this study supports Wolbachia introgression into A. Aegypti populations as a biocontrol method to reduce arboviral (e.g., DENV) transmission.