Background <p>Neonatal hypoxic-ischemic brain damage (HIBD) treatment is challenging, with lactylation potentially playing a key role. This study investigated lactylation-related genes (LRGs) in HIBD.</p> Methods <p>HIBD models used SD rats. Transcriptomics, proteomics, and scRNA-seq analyzed brain tissues across time points. Machine learning integrated DEGs/DEPs and LRG data to identify a biomarker. Inflammation (IL-1β, ELISA), oxidative stress (MDA, CAT), histopathology (HE, Nissl staining), and long-term function (Morris water maze) were assessed. Molecular docking predicted drug interactions.</p> Results <p>GFAP and LCP1 were identified as key up-regulated LRGs in HIBD, linked to ubiquitin-mediated proteolysis. Ginkgolide B and tangeretin significantly reduced acute inflammation (IL-1β), oxidative damage (MDA, CAT), improved histopathology, and enhanced long-term cognitive outcomes. scRNA-seq revealed dynamic biomarker expression during astrocyte and microglial differentiation.</p> Conclusion <p>The study defines GFAP and LCP1 as critical lactylation-associated therapeutic targets in HIBD. Ginkgolide B and tangeretin demonstrate potent neuroprotective effects, offering novel HIBD treatment strategies.</p> Impact <p><UnorderedList Mark="Bullet"> <ItemContent> <p>We explored a rat pup model of neonatal hypoxic-ischemic encephalopathy using a multi-omics approach for the first time. We also investigated the role of lactate metabolism-related genes in this model, providing potential new targets and directions for future drug development.</p> </ItemContent> </UnorderedList></p>