Abstract Juniperus pingii var. wilsonii has been traditionally used in Tibetan medicine for the treatment of inflammatory diseases. In the present study, J. pingii var. wilsonii polysaccharides (JPWP), with high content of d ‑galacturonic acid, showed potent anti-complementary activity in vitro and significantly attenuated acute lung injury (ALI) induced by H1N1 influenza virus in vivo through reducing the inflammatory responses, alleviating oxidative stress and inhibiting the activation of complement. Thus, anti-complementary activity-guided fractionation of JPWP led to the isolation of an acidic homogeneous polysaccharide, JPWP-PS, whose structure was further elucidated by acid hydrolysis, PMP derivation, methylation and NMR analysis. JPWP-PS had potent anti-complementary activity with the CH 50 value of 0.073 ± 0.009 mg/mL, and was characterized by the residues of T-Ara f -(1→, →3)-Ara f -(1→, →3,5)-Ara f -(1→, →3)-Gal p -(1→ and →4)-Gal p A-(1→. Graphical abstract Unlabelled Image Highlights • Juniperus pingii var. wilsonii polysaccharides (JPWP) improved survival rate of H1N1 virus infected mice. • JPWP treated acute lung injury via inhibiting inflammatory responses, oxidative stress and activation of complement. • A homogeneous acidic polysaccharide with potent anti-complementary activity was isolated from JPWP. [ABSTRACT FROM AUTHOR]