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The intestinal flora may be an important and modifiable factor that contributes to the immune response in influenza. To investigate the effect of intestinal flora alteration induced by antibiotic interference on microRNA (miRNA) communication in antiviral immunity, BALB/c mice received two weeks of antibiotic treatment before infection with the influenza A virus. The changes in intestinal flora and pulmonary flora were detected and analyzed by 16S ribosomal RNA (rRNA) gene sequencing. The amplification of the influenza virus in the lungs was measured by RT-PCR. The involvement of pulmonary miRNA was explored using miRNA microarray analysis. The results showed that the antibiotics destroyed the symbiotic relationship of the intestinal flora, resulting in a reduction in bacterial diversity, but they did not affect the pulmonary flora. The alteration of intestinal flora affected the expression of pulmonary miRNAs and resulted in an enhancement of pulmonary influenza virus amplification. The conclusion is that alteration of intestinal flora induced by antibiotic interference affected the expression of pulmonary miRNAs to interfere with host antiviral immunity, of which miR-146b and miR-29c might be good resources of resistance to influenza under antibiotic abuse.