Recent evidence has shown that microRNAs (miRs) are involved in endothelial dysfunction and vascular injury in lung-related diseases. However, the potential role of miR-34a in the regulation of pulmonary endothelial dysfunction, vascular injury, and endothelial cells (ECs) apoptosis in acute lung injury (ALI)/acute lung respiratory distress syndrome is largely unknown. Here, we show that miR-34a-5p was upregulated in whole lungs, isolated ECs from lungs, and ECs stimulated with various insults (LPS and hyperoxia). Overexpression of miR-34a-5p in ECs exacerbated endothelial dysfunction, inflammation, and vascular injury, whereas the suppression of miR-34a-5p expression in ECs and miR-34a–null mutant mice showed protection against LPS- and hyperoxia-induced ALI. Furthermore, we observed that miR-34a–mediated endothelial dysfunction is associated with decreased miR-34a direct-target protein, sirtuin-1, and increased p53 expression in whole lungs and ECs. Mechanistically, we show that miR-34a leads to translocation of p53 and Bax to the mitochondrial compartment with disruption of mitochondrial membrane potential to release cytochrome C into the cytosol, initiating a cascade of mitochondrial-mediated apoptosis in lungs. Collectively, these data show that downregulating miR-34a expression or modulating its target proteins may improve endothelial dysfunction and attenuate ALI.