Liver injury is a common complication of severe heat stroke (HS). Extracellular vesicles (EVs) are part of a novel pathway mediating intercellular communication. Whether EVs are involved in the pathogenesis underlying HS-induced liver injury remains unknown. Here, we explored the role of hepatocyte EVs in HS-induced liver injury and their protein regulation patterns after HS induction. Isobaric tags for relative and absolute quantification technology (iTRAQ) and liquid chromatography-tandem mass spectrometry analysis identified changes in the proteomic profiles of hepatocyte-derived heat-stroked EVs, and we identified 53 up-regulated proteins. Bioinformatics analysis verified that the regulation of programmed cell death was the most significant altered pathway. To clarify the effect of HS hepatocyte-derived EVs in inducing hepatocyte-programmed death and injury, they were added to recipient hepatocytes and injected into mice. This treatment significantly induced the synthesis of apoptosis (caspase-3/8) and necroptosis-associated proteins [receptor-interacting protein 1 (RIP1), RIP3, and mixed lineage kinase domain-like protein]; moreover, it increased the numbers of apoptotic and necroptotic cells in hepatocytes and liver tissues and increased the levels of biochemical liver injury markers (alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase). Our study is the first comprehensive analysis of the hepatocyte-derived heat-stroked EV proteome confirming the induction of liver injury by Evs. We provide a novel explanation for the pathological mechanism underlying HS-induced liver injury.