Alzheimer’s disease (AD) is the leading cause of dementia, with no effective therapies to reduce this progressive form of nerve cell death. Peptide thymosin β4 (Tβ4) controls actin polymerization and showed reduced neuroinflammation in middle stage AD model mice. To report the effect and mechanism of Tβ4 in early stage of AD model mice, Tβ4 overexpression or knockdown in 4 months APP/PS1 mice brain was achieved by genetic engineering methods. Two months later, behavioral test, electroencephalogram (EEG) test, immunofluorescence, immunohistochemistry, immunoblotting, dot blot, RT-PCR and transmission electron microscopy (TEM) were applied. The findings showed that the overexpression of Tβ4 displayed reduced microglia and astrocytes activation, arrested neuronal degeneration, repaired myelin sheath degeneration, decreased NF-κBp65 protein level, and restored EEG abnormality. Conversely, knockdown of Tβ4 led to lysosomal dysfunction, neuronal function weakening, myelin sheath loss, increased NF-κBp65 protein level, and further impaired image discrimination or spatial (working) memory in the APP/PS1 mice. The combination of TLR4 antagonist or NF-κBp65 inhibitor further reduced the inflammatory protein level. gv971 significantly reduced the glial reactivity and improved the neuronal survival, but not the learning and memory impairment of APP/PS1 mice. Combining Tβ4 with other therapies may be an effective strategy to combat the complex changes in AD.