<p>Electroacupuncture (EA) might exert endogenous protective effects on astrocytes in ischemic stroke. Nevertheless, the biological regulatory processes involved have not been identified. The astrocytes were randomly divided into six groups: the control, oxygen-glucose deprivation/reoxygenation (OGD/R), EA serum, METTL3, lncRNA MALAT1 (MALAT1) and AQP4 groups. OGD/R was performed to establish in vitro models of ischemic stroke. EA serum was obtained from rats that were received EA treatment 3 times at “Renzhong” (GV26) and “Baihui” (GV20) acupoints. The morphological characteristics of astrocytes were identified by microscopy and immunohistochemistry. Mitochondrial ultrastructure was observed using transmission electron microscopy. Cell viability and apoptosis rate were measured with cell counting kit-8 and flow cytometry, respectively. RNA m6A levels were detected by colorimetry, and the expression levels of METTL3, MALAT1 and AQP4 were tested with Western blot and quantitative real-time PCR. 10% EA serum was found to be more effective in improving astrocyte morphology and cell viability. EA serum improved mitochondrial ultrastructure, the viability and apoptosis of astrocytes in OGD/R condition, whereas overexpression of METTL3, MALAT1 and AQP4 inhibited the protective effect of EA serum on astrocytes. Furthermore, EA serum down-regulated the level of RNA m6A and the expression levels of METTL3, MALAT1 and AQP4 in OGD/R condition, while overexpression of METTL3, MALAT1 and AQP4 reversed the down-regulatory effects of EA serum. EA serum attenuates OGD/R-induced astrocyte damage in vitro, and this protective role might be achieved by down-regulating the AQP4 via m6A methylation of MALAT1.</p>