Background：Microglial phagocytosis helps regulate inflammation after ischemic stroke, but its regulation by protein lactylation remains unclear. This study investigated whether Rac1 lactylation modulates microglial function and contributes to neuroprotection.

Methods：Brain lactate was assessed using magnetic resonance spectroscopy in patients with ischemic stroke and tissue measurements in mice. Bioinformatics characterized the subcellular localization and functional characteristics of post-stroke lactylated proteins. Rac1 lactylation sites were investigated in BV2 cells and primary microglia using lactylation/delactylation models, Flag-Rac1 wild-type/mutant constructs, ¹³C₃-labeled sodium lactate, and high-performance liquid chromatography–mass spectrometry. Rac1 site-mutant mice underwent transient middle cerebral artery occlusion (tMCAO). Immunofluorescence for cleaved caspase-3, TMEM119, CD68, and LAMP1 assessed microglial apoptosis, morphology, and phagocytosis; behavioral and histopathological analyses evaluated neurological function and infarct volume.

Results：Lactate levels were elevated in patients and MCAO mice. Peri-infarct protein lactylation increased after tMCAO, correlated positively with lactate levels, and was most pronounced 24 hours after ischemia–reperfusion. Proteomic analysis identified Rac1 as a candidate. Rac1 predominantly localized to Iba1-positive microglia, with limited neuronal expression, and was lactylated in vivo and in vitro. Rac1 lactylation increased GTP-bound Rac1 and enhanced microglial phagocytosis. Rac1 site-mutant mice showed reduced Rac1 lactylation, larger infarcts, worse neurological deficits, and higher TNF, IL-6, and IL-1β expression. Mechanistic analyses suggested that lactylation reduced ubiquitination-dependent Rac1 degradation and promoted actin cytoskeletal remodeling.

Conclusions：Rac1 lactylation may confer neuroprotection by limiting Rac1 degradation, enhancing Rac1 activation, and promoting microglial phagocytosis. These findings identify Rac1 lactylation as a potential therapeutic target for ischemic brain injury.

Keywords: Rac1; Lactylation; Microglia; Ischemic stroke