Cerebral ischemia-reperfusion injury (CIRI) involves innate immunity activation in astrocytes and the inflammatory response. The interferon-induced transmembrane protein 3 (IFITM3) is an immune protein whose role in CIRI remains largely unexplored. This study investigated the role of IFITM3 in CIRI in mice. Adeno-associated virus (AAV)-mediated delivery of siRNA was used to inhibit IFITM3 expression, assessing effects on astrocyte activation, inflammatory cytokine expression. Primary cultured astrocytes were exposed to oxygen-glucose deprivation/reperfusion (OGD/R) to study IFITM3's role in vitro. Western blotting (WB) was employed to measure C-reactive protein (CRP) and IFITM3 levels, and enzyme-linked immunosorbent assay (ELISA) was used to quantify inflammatory cytokines. γ-Secretase activity, as well as Aβ40 and Aβ42 peptide levels, were measured to evaluate its activity. IFITM3 expression in astrocytes was significantly elevated following CIRI (p <
0.002), leading to increased γ-secretase activity and higher production of Aβ40 and Aβ42 peptides (p <
0.002). CRP levels were also upregulated in the context of IFITM3 expression (p <
0.01). Inhibiting IFITM3 expression via AAV-mediated delivery of siRNA significantly reduced astrocyte activation, inflammatory cytokine expression (p <
0.002 for IL-1β, IL-6, TNF-α, and IFN-γ), and improved neurobehavioral scores (p <
0.002). In vitro, IFITM3 inhibition significantly reduced the protein and mRNA levels of IFITM3, suppressed astrocyte activation, and decreased the expressions of inflammatory factors (p <
0.01). IFITM3 inhibition reduced the apoptosis of co-cultured neuronal cells (p <
0.01) and suppressed TLR4/NF-κB expression (p <
0.01), thereby attenuating the production of inflammatory factors. Inhibiting IFITM3 expression in astrocytes not only regulates γ-secretase activity but also mitigates neuroinflammation, thereby alleviating CIRI.