Abstract: Heatstroke is a heat stress–induced, life-threatening condition associated with circulatory failure and multiple organ dysfunctions. If global warming continues, heatstroke might become a more prominent cause of mortality worldwide, but its pathogenic mechanism is not well understood. We found that Z-DNA binding protein 1 (ZBP1), a Z–nucleic acid receptor, mediated heatstroke by triggering receptor-interacting protein kinase 3 (RIPK3)–dependent cell death. Heat stress increased the expression of ZBP1 through heat shock transcription factor 1 (HSF1) and activated ZBP1 through a mechanism independent of the nucleic acid sensing action. Deletion of ZBP1, RIPK3, or both mixed lineage kinase domain-like (MLKL) and caspase-8 decreased heat stress–induced circulatory failure, organ injury, and lethality. Thus, ZBP1 appears to have a second function that orchestrates host responses to heat stress.