This dataset corresponds to the DIA-based proteome analysis of Shigella flexneri. In vitro cultured S. flexneri samples were processed (cell lysis, tryptic digestion, peptide purification) and detected by data-independent acquisition liquid chromatography-tandem mass spectrometry (DIA-LC-MS/MS), with a project-specific spectral library constructed via DDA and data analyzed by DIA-NN. It includes protein identification results (UniProt IDs, sequence coverage), quantitative expression profiles (relative intensity, repeat consistency), and functional annotations (GO classification, KEGG pathway enrichment). This dataset supports differential protein screening, exploration of S. flexneri’s pathogenic mechanisms, and mining of virulence-related target proteins; it complies with PRIDE standards (including raw mass spectrometry information) for reproducible secondary analysis.