Oxidized low-density lipoprotein (ox-LDL) can impair endothelial function and lead to the atherosclerosis development. Protein S-nitrosylation is sensitive to cellular redox state and acts as a crucial regulator and executor of nitric oxide (NO) signaling pathways. Aberrant S-nitrosylation contributes to the pathogenesis of cardiovascular and cerebrovascular diseases. However, the effect of ox-LDL on S-nitrosylation and its significance for endothelial dysfunction have not been studied at proteome level. Herein, the combined quantitative analysis of proteome and S-nitrosoproteome was performed using an integrated biotin switch and iTRAQ labeling approach in EA.hy926 cell line derived from human umbilical vein endothelial cell (HUVEC) treated with ox-LDL. A total of 2204 S-nitrosylated (SNO) peptides of 1318 SNO-proteins were quantified. Notably, 352 SNO-peptides of 262 SNO-proteins were significantly regulated after excluding S-nitrosylation changes caused by protein expression alterations. Many of them belonged to mRNA splicing, ribosomal structure and translational regulatory proteins, covering the entire translation process. The results indicated that S-nitrosylation of the splicing and translational machinery in vascular endothelial cells was susceptible to ox-LDL. Abnormal protein S-nitrosylation may be one pivotal mechanism underlying endothelial dysfunction induced by ox-LDL. This study potentially enriches the present understanding of pro-atherogenic effect of ox-LDL from the perspective of S-nitrosylation. SIGNIFICANCE: The role of ox-LDL in endothelial dysfunction and atherosclerosis development has been recognized from the aspect of impaired NO production. However, its effect on S-nitrosylation, which is directly related to NO signaling pathway, still remains largely unexplored. Our work initially provided a systematic characterization of S-nitrosoproteome in ox-LDL-treated endothelial cells after ruling out the changes of S-nitrosylation modification caused by protein expression alone. MS-based approach coupled with iTRAQ technique indicated 262 SNO-proteins were significantly regulated. Functional enrichment and interaction network analysis revealed that proteins involved in mRNA splicing and translational machinery were susceptible to abnormal S-nitrosylation under ox-LDL treatment. This achievement suggested one potential mechanism underlying endothelial dysfunction induced by ox-LDL from the perspective of S-nitrosoproteome.