AIMS: Novel cancer therapies leading to increased survivorship of cancer patients have been negated by a concomitant rise in cancer therapies-related cardiovascular toxicities. Sunitinib, a first line multi-receptor tyrosine kinase inhibitor, has been reported to cause vascular dysfunction although the initiating mechanisms contributing to this side effect remain unknown. Long non-coding RNAs (lncRNAs) are emerging regulators of biological processes in endothelial cells (ECs); however, their roles in cancer therapies-related vascular toxicities remain underexplored. METHODS AND RESULTS: We performed lncRNA expression profiling to identify potential lncRNAs that are dysregulated in human-induced pluripotent stem cell-derived ECs (iPSC-ECs) treated with sunitinib. We show that the lncRNA hyaluronan synthase 2 antisense 1 (HAS2-AS1) is significantly diminished in sunitinib-treated iPSC-ECs. Sunitinib was found to down-regulate HAS2-AS1 by an epigenetic mechanism involving hypermethylation. Depletion of HAS2-AS1 recapitulated sunitinib-induced detrimental effects on iPSC-ECs, whereas CRISPR-mediated activation of HAS2-AS1 reversed sunitinib-induced dysfunction. We confirmed that HAS2-AS1 stabilizes the expression of its sense gene HAS2 via an RNA/mRNA heteroduplex formation. Knockdown of HAS2-AS1 led to reduced synthesis of hyaluronic acid (HA) and up-regulation of ADAMTS5, an enzyme involved in extracellular matrix degradation, resulting in disruption of the endothelial glycocalyx which is critical for ECs. In vivo, sunitinib-treated mice showed reduced coronary flow reserve, accompanied by a reduction in Has2os and degradation of the endothelial glycocalyx. Finally, we identified that treatment with high molecular-weight HA can prevent the deleterious effects of sunitinib both in vitro and in vivo by preserving the endothelial glycocalyx. CONCLUSIONS: Our findings highlight the importance of lncRNA-mediated regulation of the endothelial glycocalyx as an important determinant of sunitinib-induced vascular toxicity and reveal potential novel therapeutic avenues to attenuate sunitinib-induced vascular dysfunction.