The impact of low pathogenic influenza viruses such as subtype H9N2, which infect the respiratory and the gastrointestinal tracts of chickens, on microbial composition are not known. Twenty-day-old specific pathogen-free chickens were assigned to two treatment groups, control (uninfected) and H9N2-infected (challenged via the oral-nasal route). Fecal genomic DNA was extracted, and the V3-V4 regions of the 16S rRNA gene were sequenced using the Illumina Miseq® platform. Sequences were curated using Mothur as described in the MiSeq SOP. Infection of chickens with H9N2 resulted in an increase in phylum Proteobacteria, and differential enrichment with the genera Vampirovibrio, Pseudoflavonifractor, Ruminococcus, Clostridium cluster XIVb and Isobaculum while control chickens were differentially enriched with genera Novosphingobium, Sphingomonas, Bradyrhizobium and Bifidobacterium. Analysis of pre- and post-H9N2 infection of the same chickens showed that, before infection, the fecal microbiota was characterized by Lachnospiracea and Ruminococcaceae family and the genera Clostridium sensu stricto, Roseburia and Lachnospiraceae incertae sedis. However, post-H9N2 infection, class Deltaproteobacteria, orders Clostridiales and Bacteroidiales and the genus Alistipes were differentially enriched. Findings from the current study show that influenza virus infection in chickens results in the shift of the gut microbiota, and the disruption of the host-microbial homeostasis in the gut might be one of the mechanisms by which influenza virus infection is established in chickens.