MicroRNAs (miRNAs) are small non-coding RNAs that are crucial post-transcriptional regulator for host mRNAs. Recent studies indicate that miRNAs may modulate host response during RNA viruses infection. However, the role of miRNAs in immune response against H5N1 infection is not clearly understood. In this study, we show that expression of cellular miRNA, miR-324-5p was downregulated in A549 cells in response to infection with RNA viruses, H5N1, A/PR8/H1N1, and NDV and transfection with polyI:C. We found that miR-324-5p inhibited H5N1 replication by targeting the PB1 viral RNA of H5N1 in host cells. In addition, transcriptome analysis revealed that miR-324-5p enhanced the expression of Type-I, Type-III interferons, and interferons-inducible genes (ISGs) by targeting CUEDC2, the negative regulator of JAK1-STAT3 pathway. Altogether, these findings highlights that the miR-324-5p plays a crucial role in host defense against H5N1 by targeting viral PB1 and host CUEDC2 to inhibit H5N1 replication.IMPORTANCE Highly pathogenic influenza A virus (HPAIV) continues to pose a pandemic threat globally. From 2003-2017, H5N1 HPAI has caused 453 human deaths with a high mortality rate of 52.74%. This work shows that miR-324-5p suppresses the H5N1 HPAI viral replication by directly targeting viral genome (thereby inhibits viral gene expression) and cellular CUEDC2 gene, the negative regulator of interferon pathway (thereby enhance anti-viral genes). Our study enhance the knowledge of role of microRNAs in cellular response to viral infection. Also, the study provides help in understanding how the host cells utilizes small RNAs in controlling the viral burden.