The bacterial endosymbiont, Wolbachia pipientis, infects between 40-70% of all insect species. Wolbachia has previously been shown to provide protection against many pathogens including Drosophila C virus (DCV) in Drosophila and dengue virus (DENV) in Aedes aegypti. The mechanism underlying pathogen blocking is unknown. Wolbachia-initiated immune priming in novel infections has been suggested as a possible mechanism underlying pathogen blocking. However, in historically stable infections such as Drosophila, pathogen blocking does not require immune activation. It has previously been shown that in D. melanogaster, cholesterol contributes to the underlying mechanism of pathogen protection against DCV. Here we test the conservation of cholesterol’s role in pathogen protection in multiple hosts against DENV. We manipulated dietary cholesterol for Ae. aegypti and found that reduced dietary cholesterol has no effect on DENV titer in both wild type and Wolbachia-infected mosquitoes. However, when grown on diets with increased cholesterol, Wolbachia-infected mosquitoes show an increase in DENV viral copy numbers, i.e. a reduction in pathogen blocking. This viral increase is specific to Wolbachia-infected mosquitoes as wildtype mosquitoes show a reduction in DENV viral titer when on a high cholesterol diet. Furthermore, when grown on a diet with increased cholesterol, D. melanogaster infected with DENV also show reduced pathogen blocking. In both species, increased cholesterol did not alter Wolbachia densities suggesting that increased cholesterol is not reducing pathogen blocking by simply decreasing Wolbachia titers. Our results suggest that host cholesterol levels contribute to a conserved mechanism underlying Wolbachia-mediate pathogen blocking.