Detecting microproteins (miPs) translated from small open reading frames (smORFs) by mass spectrometry poses major challenges. Here we report a high-throughput proteo-genomic pipeline that identified a large number of intracellular and extracellular endothelial cell miPs encoded by non-canonical smORFs. Vascular inflammation in vitro and in vivo profoundly altered smORF expression and miPs were identified in the serum of patients following cardiac tissue damage. The functional importance of 256 miPs for endothelial cell growth and viability was demonstrated by a high-throughput CRISPR/Cas9 screen. Moreover, the inflammation-induced expression of a non-canonical miP, miP-PSTPIP2, was validated in vivo and in carotid arteries from patients with atherosclerosis. Combining gain/loss-of-function with multi-omic approaches demonstrated the involvement of miP-PSTPIP2 in the regulation of gene expression, proliferation and viability through interaction with proteins in different subcellular compartments. Thus, the proteo-genomic approach implemented here enables the identification of large numbers of miPs. Non-canonical miPs play a fundamental role in the regulation of endothelial cell function and may become biomarkers or therapeutic targets in cardiovascular disease