Background: Gastrodin (GAS) has been proven to play a therapeutic role in a variety of neurological diseases by affecting activated astrocytes, however, the underlying mechanisms have not been fully illustrated. This study aimed to investigate if GAS exerts the neuroprotective effect through regulating the Notch signaling pathway involved in reactive astrocytes. Methods: Astrocyte cell lines (TNC1 cells) were cultured in vitro. The hypoxic-ischemic cell model was prepared using the oxygen-glucose deprivation (OGD) method, GAS's pretreatment concentration was 0.34 mM, intervention for 1 hour. Cell counting kit-8 (CCK-8) assay, Transwell migration assay, immunofluorescent staining (double staining), and Western blotting were used to observe the effects of OGD or GAS interference on the function of astrocytes, and the changes of key protein expressions in the Notch signaling pathway were analyzed. Results: GAS had no obvious toxic effect on TNC1 astrocytes under physiological conditions. Following OGD, GAS can not only improve cell viability and migration, but also regulate the production of inflammatory mediators. We also found that OGD significantly increased the expression of key proteins related to the Notch signaling pathway, Notch-1, intracellular Notch receptor domain (NICD), recombining binding protein suppressor of hairless (RBP-JK), transcription factor hairy and enhancer of split-1 (Hes-1) in TNC1 astrocytes, which was significantly inhibited by GAS. In addition, GAS inhibited the OGD-induced expression of TNC1 astrocyte tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta), and enhanced the expression of nutrient factors, including brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1). The Notch signaling pathway specific inhibitor, N-[N-(3,5-Difluorophenacetyl)-1-alany1]S-phenyglycine t-butylester (DAPT), could significantly enhance the effect of GAS on TNC1 astrocytes after OGD, such as the inhibition of inflammatory factors and the up-regulation of neurotrophic factors. Conclusions: GAS exerts dual effects on astrocytes via regulation of the Notch signaling pathway. We found that it could inhibit the pro-inflammatory factors mediated by astrocytes, and also promote the secretion of neurotrophic factors by astrocytes. These results provide a new biological mechanism for the treatment of neuroinflammatory diseases by GAS.