The aim of the current study was to explore the potential role of hepatocyte exosomes in HS induced liver injury and their protein expression patterns under HS. Isobaric tags for relative and absolute quantitation technology (iTRAQ) with liquid chromatography-tandem mass spectrometry analysis were applied to explore changes in the proteomic profiles of HS-hepatocyte exosomes. Compared to the control exosome group, 53 and 21 groups of proteins were identified as up- and down-regulated in the HS-exosomes. In addition, Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway verificated that regulation of programmed cell death was the top significant altered pathway. We demonstrated in vitro and in vivo the capacity of promoting programmed liver cell death and liver injury of HS hepatocyte exosomes. This study presented the first comprehensive analysis of the HS-hepatocyte exosomal proteome, confirming the induction of HS liver injury by exosomes.