OBJECTIVE: We aimed to explore the role of SIRT3 in ameliorating neuroinflammation caused by hypoxia-ischemia (HI). METHODS: A rat model of HI was established, and 48 hours prior to constructing the HI model, the rats received an intracerebroventricular injection of a recombinant adeno-associated virus type 9 vector. TTC and Nissl staining assessed the effects of SIRT3 on cerebral infarction and brain atrophy in HI rats. Neuroinflammation was evaluated by investigating IL-1β and MPO positive cells, and ELISA for determining inflammatory cytokines. IBA-1, CD68, and iNOS positive microglia and NLRP3 activation-related protein expression were also detected. SIRT3 was overexpressed in oxygen glucose deprivation (OGD)-induced microglia model, where cell morphology and expressions of pro-inflammatory cytokines and NLRP3 inflammasome activation-related proteins were examined. Additionally, neurons co-cultured with SIRT3-overexpressing microglia were analyzed for mitochondrial damage and apoptosis. RESULTS: SIRT3 alleviated cerebral infarction and atrophy in HI rats. It also inhibited neuroinflammation, reducing IL-1β and MPO positive cells, and lowered the levels of pro-inflammatory cytokines. In both HI rat model and OGD cell model, SIRT3 inhibited excessive activation of microglia and NLRP3 inflammasome. Furthermore, co-culturing neurons with SIRT3-overexpressing microglia resulted in reduced neuronal apoptosis and improved mitochondrial function, evidenced by lower ROS levels, alleviated mitochondrial depolarization and increased ATP production. CONCLUSION: SIRT3 restrains pro-inflammatory microglia and NLRP3 inflammasome and alleviates neuroinflammation following HI brain injury.