OBJECTIVE: Ischemia-reperfusion injury can lead to brain injury by ischemia and hypoxia. Withaferin A, an analogue of metformin, shows biological function similar to that of metformin. The purpose of this study is to explore the protective mechanism of Withaferin A on brain injury from both cellular and tissue aspects. STUDY DESIGN: The rat brain cells were treated with rotenone, and the protective effect of Withaferin A on ischemia-reperfusion injury was evaluated by peroxidase activity assay, NF-kB related protein, and the detection of inflammatory factors. The release of IL-6, TNF-α was detected by ELISA kit. RESULTS: Compared with the untreated group, the death of cerebral cells and the production of ROS in cells were significantly decreased in the Withaferin A group. Meanwhile, the expression of MnSOD, catalase, and GSH-Px were significantly upregulated to reduce the damage caused by ROS, the apoptosis related protein including bax, and cleaved caspases were remarkably downregulated by the treatment of Withaferin A. NF-kB protein was obviously decreased in the Withaferin treated group. CONCLUSION: The results indicated that withaferin A can activate the antioxidant system in ischemia/ reperfusion cerebral tissues through NF-kB pathway, thus reducing the production of ROS and cell apoptosis, thus alleviating ischemia-reperfusion injury in the rat brain. (Anal Quant Cytopathol Histpathol 2021;43: 973–980) © Science Printers and Publishers, Inc.