Cell death is an important biological process that is believed to have a central role in intestinal ischaemia/reperfusion (I/R) injury. While the apoptosis inhibition is pivotal in preventing intestinal I/R, how necrotic cell death is regulated remains unknown. Necroptosis represents a newly discovered form of programmed cell death that combines the features of both apoptosis and necrosis, and it has been implicated in the development of a range of inflammatory diseases. Here, we show that receptor-interacting protein 1/3 (RIP1/3) kinase and mixed lineage kinase domain-like protein recruitment mediates necroptosis in a rat model of ischaemic intestinal injury in vivo. Furthermore, necroptosis was specifically blocked by the RIP1 kinase inhibitor necrostatin-1. In addition, the combined treatment of necrostatin-1 and the pan-caspase inhibitor Z-VAD acted synergistically to protect against intestinal I/R injury, and these two pathways can be converted to one another when one is inhibited. In vitro, necrostatin-1 pre-treatment reduced the necroptotic death of oxygen-glucose deprivation challenged intestinal epithelial cell-6 cells, which in turn dampened the production of pro-inflammatory cytokines (tumour necrosis factor-alpha and interleukin-1 beta), and suppressed high-mobility group box-1 (HMGB1) translocation from the nucleus to the cytoplasm and the subsequent release of HMGB1 into the supernatant, thus decreasing the activation of Toll-like receptor 4 and the receptor for advanced glycation end products. Collectively, our study reveals a robust RIP1/RIP3-dependent necroptosis pathway in intestinal I/R-induced intestinal injury in vivo and in vitro and suggests that the HMGB1 signalling is highly involved in this process, making it a novel therapeutic target for acute ischaemic intestinal injury.