Among the various cellular components of the liver, Kupffer cells (KCs) serve as central innate immune cells responsible for maintaining hepatic homeostasis and initiating immune responses against invading pathogens. Lysine lactylation represents a dynamic and reversible posttranslational modification, and although numerous global lactylome studies have been performed in liver and macrophage models, no such studies have been performed in LPS-stimulated KCs. In this study, we utilized global lactylome profiling to identify 1,189 lysine lactylation (Kla) sites across 671 lactylated proteins in LPS-treated immortalized mouse KCs. Distinct sequence motifs surrounding lactylated lysine residues were identified between LPS-stimulated conditions. To further characterize LPS-treated lactylated proteins, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. KEGG pathway analysis highlighted significant enrichment in pathways related to spliceosome and neutrophil extracellular traps (NETs). In particular, we identified lactylation sites on key immune regulatory proteins, including HMGB1 and HDAC, which are closely related to NET formation. In addition, we identified a novel lactylation site at lysine 82 (K82) of HMGB1 in a previous study. These findings highlight the important role of lysine lactylation in regulating Kupffer cell function, particularly in the context of LPS-induced inflammatory responses. The identification of lactylation sites on key regulatory proteins, such as HMGB1 and HDAC, suggests a potential mechanistic link between these modifications and immune regulation, including NETs.