Obligatory blood-triggered reproductive strategy is an evolutionary adaptation of mosquitoes for rapid egg development. It contributes to the vectorial capacity of these insects. Therefore， understanding the molecular mechanisms underlying reproductive processes is of particular importance. Here， we report that microRNA-309 (miR-309) plays a critical role in mosquito reproduction. A spatiotemporal expression profile of miR-309 displayed its blood feeding-dependent onset and ovary-specific manifestation in female Aedes aegypti mosquitoes. Antagomir silencing of miR-309 impaired ovarian development and resulted in nonsynchronized follicle growth. Furthermore， the genetic disruption of miR-309 by CRISPR/Cas9 system led to the developmental failure of primary follicle formation. Examination of genomic responses to miR-309 depletion revealed that several pathways associated with ovarian development are down-regulated. Comparative analysis of genes obtained from the high-throughput RNA sequencing of ovarian tissue from the miR-309 antagomir-silenced mosquitoes with those from the in silico computation target prediction identified that the gene-encoding SIX homeobox 4 protein (SIX4) is a putative target of miR-309. Reporter assay and RNA immunoprecipitation confirmed that SIX4 is a direct target of miR-309. RNA interference of SIX4 was able to rescue phenotypic manifestations caused by miR-309 depletion. Thus， miR-309 plays a critical role in mosquito reproduction by targeting SIX4 in the ovary and serves as a regulatory switch permitting a stage-specific degradation of the ovarian SIX4 mRNA. In turn， this microRNA (miRNA)-targeted degradation is required for appropriate initiation of a blood feeding-triggered phase of ovarian development， highlighting involvement of this miRNA in mosquito reproduction.