Gut-lung axis injury is a common finding in patients with respiratory diseases as well as in animal model of influenza virus infection. Influenza virus damages the intestinal microecology while affecting the lungs. Rifaximin, a non-absorbable derivative of rifamycin, is an effective antibiotic that acts by inhibiting bacterial RNA synthesis. This study aimed to determine whether rifaximin-perturbation of the intestinal microbiome leads to protective effects against influenza infection, via the gut-lung axis. Our results showed that influenza virus infection caused inflammation of and damage to the lungs. The expression of tight junction proteins in the lung and colon of H1N1 infected mice decreased significantly, attesting that the barrier structure of the lung and colon was damaged. Due to this perturbation in the gut-lung axis, the intestinal microbiota became imbalanced as Escherichia coli bacteria replicated opportunistically, causing intestinal injury. When influenza infection was treated with rifamixin, qPCR results from the gut showed significant increases in Lactobacillus and Bifidobacterium populations, while Escherichia coli populations markedly decreased. Furthermore, pathology sections and western blotting results illustrated that rifaximin treatment strengthened the physical barriers of the lung-gut axis through increased expression of tight junction protein in the colon and lungs. These results indicated that rifaximin ameliorated lung and intestine injury induced by influenza virus infection. The mechanisms identified were the regulation of gut flora balance and intestinal and lung permeability, which might be related to the regulation of the gut-lung axis. Rifaximin might be useful as a co-treatment drug for the prevention of influenza virus infection.