AbstractAe. aegypti mosquitoes transmit some of the most important human viral diseases that are responsible for a significant public health burden worldwide. The small interfering RNA (siRNA) pathway is considered the major antiviral defense system in insects. Here we show that siRNA pathway disruption by CRISPR/Cas9-based Ago2 knockout impaired the mosquitoes’ ability to degrade arbovirus RNA leading to hyper-infection accompanied by cell lysis and tissue damage. Ago2 disruption impaired DNA repair mechanisms and the autophagy pathway by altering histone abundance. This compromised DNA repair and removal of damaged cellular organelles and dysfunctional aggregates promoted mosquito death. We also report that hyper-infection of Ago2 knockout mosquitoes stimulated a broad-spectrum antiviral immunity, including apoptosis, which may counteract infection. Taken together, our studies reveal novel roles for Ago2 in protecting mosquitoes from arbovirus infection and associated death.