Rationale: MicroRNAs (miRNAs) are key regulators of vascular development and diseases. The function and underlying mechanism of endothelial miRNAs have not been fully defined. Objective: To investigate the role of endothelial miR-126 in zebrafish vascular development. Methods and Results: Two homologs of miR-126, miR-126a (namely miR-126 in previous literature) and miR-126b, with only 1 nucleotide difference in their mature sequences, were identified in zebrafish genome. In vitro analysis showed that both precursors could sufficiently produce mature functional miRNAs. Expression analyses by Northern blot and quantitative RT-PCR showed that both miR-126s accumulated significantly 12 hours after fertilization and were specifically expressed in endothelial cells of zebrafish. Inhibition of miR-126a or miR-126b with specific morpholinos caused cranial hemorrhage, and simultaneous inhibition of both miR-126s resulted in a pronounced hemorrhage in higher percentage of embryos. Bioinformatics prediction showed that the targets of miR-126a/b partially overlapped but essentially differed. p21-activated kinase1 (pak1) was identified as a novel target of miR-126a/b, and pak1 3' untranslated region was differently regulated by these 2 miRNAs. Quantitative RT-PCR, in situ hybridization, and Western blot analyses showed that the level of pak1 was reduced when miR-126a/b were overexpressed. Notably, pak1 expression in endothelial cells was increased when miR-126a/b were knocked down. Furthermore, overexpression of the active form of human pak1 caused cranial hemorrhage, and knockdown pak1 effectively rescued the hemorrhage caused by inhibiting miR-126a/b. Conclusions: Two functional endothelial cell-specific miRNAs, miR-126a and miR-126b, synergistically regulate zebrafish vascular integrity, and pak1 is a critical target of miR-126a/b in vascular development.