This study performs 4D Smart DIA quantitative proteomic analysis on lysosomal fractions isolated from porcine alveolar macrophages (PAMs) that were first infected with African swine fever virus (ASFV) and then subjected to bacterial challenge. Lysosomal extraction was carried out at 6 h post‑bacterial challenge, and the resulting proteomic data are used to delineate the proteomic reprogramming of lysosomes during ASFV infection and to elucidate the roles of lysosomes in host‑virus interactions. PAMs were initially infected with ASFV, followed by bacterial stimulation. After bacterial challenge, cells were harvested and lysosomal fractions were isolated by differential centrifugation. Proteins were extracted in 8 M urea buffer supplemented with protease inhibitors, then processed through dithiothreitol reduction, iodoacetamide alkylation, and overnight trypsin digestion without HPLC prefractionation. Peptide separation was conducted on a NanoElute UHPLC system with a 15‑min linear gradient (6%–80% mobile phase B) at 500 nL/min, followed by mass spectrometric detection on a timsTOF Pro 2 instrument in dia‑PASEF mode. MS1 spectra were acquired from m/z 300 to 1500, with 20 PASEF MS/MS scans covering sequential 7 m/z windows across m/z 400–850. For bioinformatic processing, all identified proteins were annotated via Gene Ontology, KEGG pathway, Pfam domain, subcellular localization and COG/KOG classification. Differentially expressed proteins between infected and control groups were further subjected to functional enrichment analysis, hierarchical clustering of enrichment profiles, and protein‑protein interaction network construction. This project generates a systematic lysosomal proteomic dataset of ASFV‑infected and bacterially challenged PAMs, which lays a quantitative proteomic foundation for deciphering ASFV pathogenesis and lysosome‑associated host immune responses.