Secondary bacterial infection is the main cause of pneumonia after influenza virus infection. This study aims to explore the impact of co-infection of influenza A virus (IAV) and Streptococcus pneumoniae (SP) on lung immune response using a human lung-on-chip model and single-cell RNA sequencing technology, with a focus on the molecular structure and mechanism of action of the tight junction protein ZO-1 in this process. Research and construct a human lung-on-chip model to simulate the microenvironment of the lungs in vivo. Use this model to infect IAV and SP separately, as well as co-infect both. Using single-cell RNA sequencing technology to analyze gene expression profiles of lung cells under different infection states, and interpreting key pathways through GO enrichment analysis. Separate peripheral blood mononuclear cells and perform macrophage differentiation, using multiple bead immunoassay to detect cytokine levels. Evaluate changes in lung barrier function through immunofluorescence staining and image analysis. Single cell RNA sequencing data revealed unique transcriptome changes induced by IAV and SP co-infection, particularly in lung epithelial cells. The results showed that co-infection significantly downregulated the expression of tight junction protein ZO-1 and affected its intracellular localization, thereby disrupting the integrity of the pulmonary epithelial barrier. GO enrichment analysis further elucidated the signaling pathways and biological processes associated with ZO-1 downregulation. Multiple bead immunoassay showed that co-infection led to an increase in the release of specific cytokines. The study utilized a human lung-on-chip model and single-cell RNA sequencing technology to reveal the complex immune response induced by IAV and SP co-infection, and identified the key role of ZO-1 in maintaining lung barrier integrity. The downregulation and abnormal localization of ZO-1 expression may be a key mechanism leading to lung injury after co-infection.