<p>Recombinant human interferon-α1b (rhIFN-α1b) has shown proven antiviral and immunomodulatory effects in multiple viral infections. This study was designed to explore the protective effect and mechanism of rhIFN-α1b on lung injury in mice infected with influenza A virus (H1N1). The lung-adapted strain of influenza A virus NYMCX-179&#xa0;A was inoculated into mice via intranasal infection, followed by administration of rhIFN-α1b. Mouse lung tissues exhibited significant inflammatory damage, enhanced HA expression, and distinct distribution of viral particles (observed by TEM). Administration of rhIFN-α1b or oseltamivir reduced mouse mortality, alleviated lung tissue inflammation, downregulated HA expression, decreased H1 mRNA levels, and attenuated the secretion of inflammatory cytokines. Moreover, the in vivo protective effect of rhIFN-α1b administered via nebulization was stronger than that of intramuscular injection. In vitro, rhIFN-α1b reduced inflammatory cytokine secretion, diminished HA protein and H1 mRNA levels, and inhibited apoptosis in H1N1 virus-infected MDCK cells. Mechanistically, rhIFN-α1b increased the protein levels of phosphorylated JAK2 (p-JAK2) and phosphorylated STAT3 (p-STAT3) in both H1N1 virus-infected MDCK cells and lung tissues from mice with lung injury. Additionally, treatment with AG490—a JAK/STAT signaling inhibitor—reversed the effects of rhIFN-α1b on HA expression, inflammatory cytokine secretion, and cell apoptosis in H1N1 virus-infected MDCK cells. These findings demonstrated that rhIFN-α1b ameliorates lung tissue damage in H influenza A virus (H1N1)-infected mice, with the protective effect of nebulized rhIFN-α1b being stronger than that of intramuscularly administered rhIFN-α1b. Furthermore, the protective effect of rhIFN-α1b is associated with the activation of the JAK/STAT signaling pathway.</p>