The influenza A virus poses serious public health challenges worldwide. Strikingly, small noncoding microRNAs (miRNAs) that modulate gene expression are closely involved in antiviral responses, although the underlying mechanisms are essentially unknown. We now report that microRNA-340 (miR340) is downregulated following influenza A and other RNA virus infections, implying that host cells deplete miR340 as an antiviral defense mechanism. Accordingly, the inhibition or knockdown of endogenous miR340 clearly prevents the infection of cultured cells, whereas the forced expression of miR340 significantly enhances virus replication. Using next-generation sequencing, we found that miR340 attenuates cellular antiviral immunity. Moreover, mechanistic studies defined miR340 as a repressor of RIG-I and OAS2, critical factors for the establishment of an antiviral response. Collectively, these data indicate that host cells may lower their viral loads by regulating miRNA pathways, which may, in turn, provide new opportunities for treatment.