The dysfunctional immune response and multiple organ injury in sepsis is a recurrent theme impacting prognosis and mortality, while the lung is the first organ invaded by sepsis. To systematically elucidate the transcriptomic changes in the main constituent cells of sepsis-injured lung tissue, we applied single-cell RNA sequencing to the lung tissue samples from septic and control mice and created a comprehensive cellular landscape with 25044 cells, including 11317 immune and 13727 non-immune cells. Sepsis alters the composition of all cellular compartments, particularly neutrophils, monocytes, T cells, endothelial, and fibroblasts populations. Our study firstly provides a single-cell view of cellular changes in septic lung injury. Furthermore, by integrating bulk sequencing data and single-cell data with the Scissors-method, we identified the cell subpopulations that are most associated with septic lung injury phenotype. The phenotypic-related cell subpopulations identified by Scissors-method were consistent with the cell subpopulations with significant composition changes. The function analysis of the differentially expressed genes (DEGs) and the cell-cell interaction analysis further reveal the important role of these phenotype-related subpopulations in septic lung injury. Our research provides a rich resource for understanding cellular changes and provides insights into the contributions of specific cell types to the biological processes that take place during sepsis-induced lung injury.