Background. Alveolar type II (ATII) epithelial cells are the primary site of influenza virus replication in the distal lung. Development of acute respiratory distress syndrome (ARDS) in influenza-infected mice correlates with significant alterations in ATII cell function. However, the impact of infection on ATII cell surfactant lipid metabolism has never been explored. Methods. C57BL/6 mice were inoculated intranasally with 10,000 pfu/mouse of influenza A/WSN/33 (H1N1) or mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 2 and 6 days post-infection. Levels of 77 surfactant lipid-related compounds of known identity in each ATII cell sample were measured by UHPLC/MS. In other mice, bronchoalveolar lavage fluid was collected to measure lipid and protein content using commercial assay kits. Results. Relative to mock-infected animals, ATII cells from influenza-infected mice contained reduced levels of major surfactant phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) but increased levels of minor phospholipids (phosphatidylserine, phosphatidylinositol, and sphingomyelin), cholesterol, and diacylglycerol. These changes were accompanied by reductions in cytidine 5'diphospho (CDP)-choline and CDP-ethanolamine (liponucleotide precursors for ATII cell phosphatidylcholine and phosphatidylethanolamine synthesis, respectively). ATII cell lamellar bodies were ultrastructurally abnormal after infection. Changes in ATII cell phospholipids were reflected in the composition of bronchoalveolar lavage fluid, which contained reduced amounts of phosphatidylcholine and phosphatidylglycerol but increased sphingomyelin, cholesterol, and protein. Conclusions. Influenza infection significantly alters ATII cell surfactant lipid metabolism, which may contribute to surfactant dysfunction and development of ARDS in influenza-infected mice.