Long non-coding RNAs (lncRNA) serve an important role in neonatal hypoxic-ischemic encephalopathy (HIE) have been reported to regulate the activity of HIE-associated proteins. The present study aimed to elucidate the role of Hox transcript antisense intergenic RNA (HOTAIR) in oxygen-glucose deprivation/reperfusion (OGD/R)-induced injury in human brain microvascular endothelial cells (hBMVECs). The levels of HOTAIR were evaluated in the serum of neonatal patients with HIE, and the effects of HOTAIR were evaluated using in vitro assays, such as reverse transcription-quantitative PCR to detect lncRNA and mRNA levels and western blot analysis to determine protein levels. Moreover, RNA immunoprecipitation assays were used to evaluate the association between HOTAIR and enhancer of zeste homolog 2 (EZH2), Cell Counting Kit-8 was used to detect cell viability, an endothelial monolayer cell permeability assay was used to analyze cell viability, TUNEL staining was used to detect the levels of apoptosis, a Transwell assay was used to evaluate cell invasion and a tube formation assay was used to analyze tube formation ability. In addition, the effects of HOTAIR and EZH2 on cell apoptosis and the invasive and tube formation abilities of hBMVECs were investigated using TUNEL, Transwell and tube formation assays, respectively. The results showed that the expression levels of HOTAIR were markedly increased both in neonatal HIE patients and in the OGD/R injury in vitro model. HOTAIR knockdown reduced hBMVEC viability, enhanced cell permeability and apoptosis, in addition to decreasing the expression levels of tight junction-related proteins, such as zonula occludens-1, occluden, Claudin5 and vascular endothelial-cadherin. However, EZH2 overexpression reversed the effects of HOTAIR silencing on hBMVECs. Additionally, HOTAIR knockdown enhanced the migratory and tube formation abilities of OGD/R-induced hBMVECs, which were also reversed by EZH2 overexpression. Overall, the present study revealed an association between the HOTAIR/EZH2 axis and brain microvascular endothelial cell injury and angiogenesis, which provides a novel insight into the molecular mechanism underlying stroke or the development of new pharmacotherapies.