This study focused on the chemotactic activity of macrophages and the role of the TLR9 signaling pathway in the pathogenesis of viral Acute Lung Injury (ALI). For this purpose, a total of 40 male SPF mice were used, aged 5-8 weeks. They were randomly divided into an experimental group and a control group. The experimental group was further divided into S1 and S2, and the control group was further divided into D1 and D2, with 10 in each. The different groups were detected for the expression of inflammatory cytokines and chemokines and the expression of alveolar macrophages. Results showed that as for the weight, survival status, arterial blood gas analysis, lung index and wet-to-dry value of lung tissue, and lung histopathological analysis results, the S2 group showed more obvious changes versus the D2 group, and the difference was statistically significant (P<0.05). S2 had higher levels of the inflammatory factors TNF-α, IL-1β, IL-6 and the chemokine CCL3 in the BALF supernatant versus the D2 Group, and the difference is statistically significant (P<0.05). S2 had higher expression levels of chemokines CCR5, TLR9, and JMJD1A mRNA versus the D2 group, and the difference was statistically significant (P<0.05). In conclusion, the establishment of a mouse ALI model induced by poly l:C was successful; AM has a certain chemotactic activity on CCL3; polyI:C can promote the expression activity and chemotactic activity of macrophages CCR5 through signal pathways, such as TLR9.