This study aims to explore the effects and potential mechanisms of PKM2-mediated neuroinflammation leading to mitochondrial damage and its role in the progression of chronic fatigue syndrome (CFS). Bioinformatics methods were applied to predict and analyze PKM2 and downstream signaling factors. In vivo experiments were conducted with mice divided into four groups after different treatments: control group, model group, Model + PKM2-OE group, and Model + PKM2-KD group. Morris water maze and field tests were used to assess cognitive function, grip strength, and rotation tests to evaluate physical strength. HE and Nissl staining were used to observe cellular conditions in the CA1 region of the hippocampus. Immunohistochemistry was used to detect PKM2 levels in the CA1 region. Western blot was performed to assess protein expression, lactate assay kits measured serum and brain tissue lactate levels, and ELISA detected inflammatory factors in brain tissue. Bioinformatics analysis showed that PKM2 could promote the expression of glycolytic factors, leading to H4K12la histone lactylation modification, which enhances the expression of inflammatory factors such as NF-κB, resulting in mitochondrial damage. Compared to the control group, the cognitive function of the model group significantly declined, while the cognitive function of the Model + PKM2-OE group improved. However, cognitive function worsened in the Model + PKM2-KD group compared to the model group. The physical strength of the control group was normal, and no significant differences were observed in the model, Model + PKM2-OE, and Model + PKM2-KD groups. Cell quantity and arrangement in the control group were normal, while the model group showed fewer and disorganized cells. The Model + PKM2-OE group showed further deterioration compared to the model group, whereas the Model + PKM2-KD group showed improvement. Compared to the control group, the model group had increased expression of PKM2, H4K12la, H4, IL-1β, and TNFα. Compared to the model group, these markers were even higher in the Model + PKM2-OE group, but significantly reduced in the Model + PKM2-KD group. Serum lactate levels increased in the model group compared to the control group, but there was no significant difference between the Model + PKM2-OE and Model + PKM2-KD groups. Brain tissue lactate levels increased in the model group, further elevated in the Model + PKM2-OE group, but decreased in the Model + PKM2-KD group. PKM2 in hippocampal cells enhances glycolysis, lactate accumulation, and H4K12la/NF-κB-mediated neuroinflammation, leading to mitochondrial damage and accelerating the progression of chronic fatigue syndrome.© 2025. The Author(s).