Chronic lung diseases are among the major causes of death and disease worldwide. Pulmonary hypoxia is a common complication of chronic lung diseases leading to the development of pulmonary hypertension. The underlying sustained increase in vascular resistance in hypoxia is a response unique to the lung, suggesting that there are genes whose expression is selectively modulated in the lung. MicroRNA (miRNA) modulation of gene expression is emerging as an important regulatory mechanism in human disease. The aim of the study was to identify the miRNA profile underlying lung selective gene expression in hypoxia. Primary human microvascular endothelial cells from lung and cardiac tissue were cultured in normoxia or hypoxia (1% O2) for 3hr, 24hr or 48hrs (n=6 experiments for each time-point). Total RNA was extracted using the mirVana RNA Isolation kit (ABI, USA) and hypoxic conditions confirmed by TaqMan analysis using the hypoxic responsive gene VEGF-A. miRNA microarrays (n=48; LC Sciences-AS1001), which allow the simultaneous analysis of 1,719 human miRNAs, were used. RNA was reverse-transcribed to cDNA using Superscript II RNase H-Reverse Transcriptase kit (Invitrogen, UK) or Taqman microRNA Reverse Transcriptase kit (ABI, USA). Hypoxic conditions were confirmed by Taqman analysis using hsa-miR-210 (ABI, USA). The Eukaryotic 18S rRNA and RNU6B were used as endogenous controls (ABI, USA). Using a subtractive miRNA strategy, a cohort of 238 miRNA probes were identified which were differentially regulated in response to hypoxia in the pulmonary (p<0.05), but not the cardiac cells. Of these, 227 miRNAs were uniquely altered in the lung endothelium only and included miR-424 (previously reported as up-regulated in hypoxic human endothelial cells). Nine miRNAs were down-regulated in the lung and up-regulated in the heart, while two miRNAs (miR-18b and miR-19b) were up-regulated in the lung and down-regulated in the heart. Our subtractive approach also revealed 7 miRNAs that were up-regulated in the lung cells > 2-fold but showed no significant increase of expression in cardiac cells (miR-10b, miR-19b, miR-30b, miR-125a-5p, miR-20b, miR-466 and miR-568). These findings supported the hypothesis that miRNA expression was modulated by hypoxia in the pulmonary endothelium in a manner that was specific to that cell type and was different from the pattern of gene response observed in cardiac endothelial cells. Little has been reported of many of the hypoxic responsive lung-selective miRNAs that were identified in the microarray, highlighting novel targets for in-depth studies in our future work. Further analysis of miRNA/mRNA targets could reveal a role for these miRNAs in hypoxic lung disease, suggesting that therapeutic manipulation of these miRNAs may represent novel treatment strategies.