Melanin formation has a significant influence on mosquito vector competence by limiting the development of metazoan parasites. Tyrosine, the rate-limiting substrate of melanin production, can be obtained exogenously or derived from phenylalanine by phenylalanine hydroxylase (PAH). The characteristics of this defense mechanism, such as temporal expression of constituent enzymes involved in the biosynthetic pathway, can vary considerably between mosquito species. We investigated the functional role of PAH in the melanotic encapsulation response in Aedes aegypti and Armigeres subalbatus, two mosquito species with markedly different melanization responses. We used double-stranded RNA (dsRNA) to knock down PAH and observed the phenotypic effects on melanin formation. PAH transcripts were dramatically reduced in both mosquito species after gene knock down. The abundance of PAH proteins was decreased in gene knockdown mosquitoes that were inoculated with Dirofilaria immitis microfilariae (mf) as compared to inoculation controls. A significant reduction of mf melanization also was observed in these knockdown mosquitoes as compared to inoculation controls. Our data suggest that PAH is required for a fully functional melanotic encapsulation response in both mosquito vectors.