This study aimed to examine whether Biliverdin, which is a common metabolite of haem, can alleviate cerebral ischemia reperfusion injury (CIRI) by inhibiting pyroptosis. Here, CIRI was induced by middle cerebral artery occlusion-reperfusion (MCAO/R) in C57BL/6 J mice and modelled by oxygen and glucose deprivation/reoxygenation (OGD/R) in HT22 cells, it was treated with or without Biliverdin. The spatiotemporal expression of GSDMD-N and infarction volumes were assessed by immunofluorescence staining and triphenyltetrazolium chloride (TTC), respectively. The NLRP3/Caspase-1/GSDMD pathway, which is central to the pyroptosis process, as well as the expression of Nrf2, A20, and eEF1A2 were determined by Western-blots. Nrf2, A20, and eEF1A2 interactions were verified using dual-luciferase reporter assays, chromatin immunoprecipitation, or co-immunoprecipitation. Additionally, the role of Nrf2/A20/eEF1A2 axis in modulating the neuroprotective properties of Biliverdin was investigated using A20 or eEF1A2 gene interference (overexpression and/or silencing). 40 mg/kg of Biliverdin could significantly alleviate CIRI both in vivo and in vitro, promoted the activation of Nrf2, elevated A20 expression, but decreased eEF1A2 expression. Nrf2 can bind to the promoter of A20, thereby transcriptionally regulating the expression of A20. A20 can furthermore interacted with eEF1A2 through its ZnF4 domain to ubiquitinate and degrade it, leading to the downregulation of eEF1A2. Our studies have also demonstrated that either the knock-down of A20 or over-expression of eEF1A2 blunted the protective effect of Biliverdin. Rescue experiments further confirmed that Biliverdin could regulate the NF-κB pathway via the Nrf2/A20/eEF1A2 axis. In summary, our study demonstrates that Biliverdin ameliorates CIRI by inhibiting the NF-κB pathway via the Nrf2/A20/eEF1A2 axis. Our findings can help identify novel therapeutic targets for the treatment of CIRI.Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.