Vector innate immunity is an important component of determining vector competence for arboviruses. We are defining molecular pathways that mosquitoes use to limit arboviral dissemination and characterizing real-time alphavirus/mosquito interactions by employing recombinant fluorescent protein-producing alphaviruses. We have shown that the RNAi pathway proteins Dicer-2 and Argonaute-2 are key in limiting the spread of O’nyong-nyong virus throughout Anopheles gambiae mosquitoes and of Sindbis virus throughout Aedes aegypti and Culex spp. mosquitoes. Artificial silencing of these proteins through the administration of exogenous double-stranded RNAs causes significant increases in viral replication and dissemination in these mosquitoes. Furthermore, silencing of the RNA interference pathway also causes significantly higher mortality in virus-fed adults presumably because virus spreads unchecked through these mosquitoes as compared with controls. Natural fluctuations in Dicer-2, Argonaute-2, and Tudor SN transcipts were also assessed by quantitative PCR after oral infection with one of four viruses representing three arbovirus families. Most transcripts exhibited variable fluctuations; however, Dengue-2 virus significantly induced Dicer-2 and Argonaute-2 transcription in Aedes aegypti midguts 1 day post-infection. Our data demonstrates that RNA interference is a primary innate immune defense employed by many mosquitoes against alphaviruses.