To investigate the replication of influenza A virus A/Puerto Rico/8/34 (H1N1) in pulmonary microvascular endothelial cells and its effect on endothelial barrier function.Human pulmonary microvascular endothelial cells were infected with influenza A/Puerto Rico/8/34 (H1N1) virus. Plaque reduction assay, real-time quantitative PCR, immunofluorescence staining, and western blot were used to elucidate the replication process of virus-infected endothelial cells. In addition, real-time quantitative PCR was used to detect the relative expression levels of mRNA of some inflammatory factors. The endothelial resistance assay was used to determine the permeability of the endothelial monolayer. Excavation and analysis of data from open databases, such as the GeneCards database, DAVID Bioinformatics Resources, STRING search tool, and DGIdb database determined the genes, proteins, and signal pathways related to microvascular leakage caused by the H1N1 virus, and predicted the drugs that could be effective for treatment.In vitro experiments showed that the influenza virus can infect endothelial cells, leading to a significant increase in the permeability of pulmonary microvascular endothelial cells and the release of pro-inflammatory cytokines, but does not efficiently replicate in endothelial cells. A total of 107 disease-related target genes were obtained from the Gene-cards database. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that these genes mainly affected the pathways related to "Inflammatory bowel disease" (IBD), "Chagas disease" (American trypanosomiasis), "Influenza A", and also played a key role in anti-inflammation and regulation of immunity. After enrichment analysis, 46 hub genes were screened. A total of 42 FDA-approved drugs corresponding to the hub genes were screened from the DGIdb database, and these could be formulated for topical application. In addition, these drugs can be used to treat other diseases, including cancer, inflammatory diseases, immune system disorders, and cardiovascular diseases.H1N1 influenza virus affects the barrier function of endothelial cells indirectly. Combined with bioinformatics tools, we can better understand the possible mechanism of action of influenza A (H1N1) virus causing pulmonary microvascular leakage and provide new clues for the treatment of pulmonary microvascular leakage.©2021 Han et al.