Abstract: Objective To study the protein markers related to oxidative damage involved in the human artery endothelial cells injury induced by crotonaldehyde using tandem mass tag（TMT）-based liquid chromatography-tandem mass spectrometry（LC-MS/MS） and bioinformatics analysis. Methods The concentration of crotonaldehyde required to cause 90% cell damage(LD₉₀) was determined according to the cell viability damage curve. Proteomic analysis was used to detect the protein expression in crotonaldehyde-treated and untreated human arterial endothelial cells. The data on proteins which related with oxidative stress were analyzed by bioinformatics and confirmed by parallel reaction monitoring. The effect of target protein changes on oxidative function was confirmed by detecting mitochondrial membrane potential and measuring glutathione activity by spectrophotometry. Results A total of 153 significantly differentially expressed proteins were determined according to the criterion of multiple change＞1.5, in which 12 were related to oxidative damage: sensor protein（A0A2P9AJA0）, activating signal cointegrator 1 complex subunit 2（B1AH59）, nicotinamide mononucleotide adenylyltransferase 1（B1AN62）, cDNA FLJ58404（B4DRL9）, cDNA FLJ55250（B4DV58）, cDNA FLJ55007（B4DY35）, oxidase（cytochrome C） assembly 1-like（OXA1L, E7EVY0）, electron transfer flavoprotein subunit alpha（H0YL12）, cytochrome C oxidase subunit VIa homolog（O95101）, Glutamate-cysteine ligase regulatory subunit（P48507） and cytochrome C oxidase assembly factor 4 homolog（Q9NYJ1）, 39S ribosomal protein L3（P09001）. The gene ontology（GO） analysis and Kyoto encyclopedia of genes and genomes（KEGG） analysis showed that the biological process of differentially expressed proteins was related to the metabolism of carbohydrates, fatty acids and other organic substances, while the metabolic process was closely related to the production of ATP, the production and aggregation of metabolic oxidation products, and the interaction of nicotinamide adenine dinucleotide（NAD） or nicotinamide adenine dinucleotide phosphate（NADP）. Exposure to crotonaldehyde was found to be linked to reduced mitochondrial membrane potential and glutathione activity. Protein-protein interaction network analyses showed that OXA1L, electron transfer flavoprotein subunit alpha（ETFA）, mitochondrial ribosomal protein L3（MRPL3） and glutamate-cysteine ligase modifier subunit（GCLM） were the four key protein biomarkers. Conclusion Crotonaldehyde significantly changes the protein expression pattern of human artery endothelial cells which associated with the oxidative stress, which is characterized by increased expression of OXA1L, ETFA, GCLM proteins and decreased expression of MRPL3.