Pulmonary hypoxia is a common complication of chronic lung diseases leading to the development of pulmonary hypertension. The underlying sustained increase in vascular resistance is a response unique to the lung, suggesting that there are genes whose expression is selectively modulated in the lung in hypoxia. Recent studies suggest an important role for microRNAs (miRNAs) in hypoxia-mediated responses. miRNAs are short RNA sequences that modulate gene expression. The aim of the present study was to elucidate the miRNA profile underlying lung-selective gene expression in hypoxia. Primary human microvascular endothelial cells from lung (HMVEC-L) and cardiac (HMVEC-C) cells were cultured in normoxia or hypoxia (1% O2) for 3hr, 24hr or 48hrs (n=6 experiments for each time-point). To identify lung-selective miRNAs, total RNA was extracted and hypoxic conditions confirmed by real time PCR using the hypoxic responsive gene VEGF-A. RNA was probed to miRNA microarrays (MRA-1001; 1,719 human miRNAs), and results confirmed by real time PCR analysis. In silico analysis using TargetScan and microRNA.org identified genes targeted by identified miRNAs. The predicted gene targets were confirmed by real time PCR. Using a subtractive miRNA strategy, 238 lung-selective hypoxic responsive miRNAs were identified (ANOVA p<0.05) which were differentially regulated in response to hypoxia in the pulmonary (p<0.05), but not the cardiac cells. Confirmatory real time PCR experiments showed that miR-125a-5p and miR-424 were significantly up-regulated in response to hypoxia in vitro. In silico analysis predicted that miR-424 targets aquaporin 11 (AQP11). Aquaporins are water channels that have been documented to transport gases such as the vasodilator Nitric Oxide. Furthermore, miR-424 targets cullin 2 (CUL2), which has previously been shown to stabilize hypoxia-inducible factor-α and promote angiogenesis. In line with miRNA inhibiting expression of their target genes, real time PCR confirmed that both AQP11 and CUL2 were down-regulated in response to hypoxia in HMVEC-L. We conclude that hypoxia, causes alterations in miRNA expression in the pulmonary endothelium in a manner that is specific to that cell type and is different from the pattern of gene response observed in cardiac endothelial cells. We have identified a number of miRNAs which are potentially important mediators of vascular changes in hypoxic lungs disease. Little has been reported of many of the hypoxic responsive lung-selective miRNAs identified in our microarray, however, based on current knowledge of gene function, miR-424 may play important roles in pulmonary vascular remodelling and angiogenesis.